1,013 research outputs found
Bridging the Gaps: Investigating Molecular Mechanisms That Coordinate Actin Filament Assembly
No full textCell division, migration, and maintaining cell morphology are all essential and dynamic cell processes that require precise coordination of the actin cytoskeleton. Actin monomers assemble into polar actin filaments that have a faster-growing (barbed) end and a slower-growing (minus) end. Here we examine the role of IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in regulating actin filament assembly. This 189 kDa actin- binding protein slows actin filament assembly by interacting with the barbed end. Using extensive truncation analysis and single molecule microscopy techniques, we determined that IQGAP1 interacts with actin filament ends via residues within its IQ motifs. The barbed ends of actin filaments are intricately and competitively regulated by specific proteins and complexes of proteins that promote (formins) or inhibit (capping proteins) actin filament growth. We next examined the role of IQGAP1 in competitive interactions with the prominent barbed end regulators including formin and capping protein. Using fluorescently tagged proteins, IQGAP1 can be directly visualized on filament ends with individual formins and capping proteins and with formin-capping protein complexes. Interactions between IQGAP1 and formin on the ends of filaments slows formin-mediated actin assembly from 22.68 ± 2.9 subunits s-1μM-1 to 6.13 ± 0.7 subunits s-1μM-1. Further, IQGAP1 interacts with decision complexes on filament ends, creating a more complex decision complex, which decreases the dwell time on the end by 18-fold. We next examined the relevance of IQGAP1-mediated capping in cells using readouts of actin assembly: cell morphology, actin filament structure, and cell migration. Cells lacking IQGAP1 displayed significant changes to cell morphology and actin filament structures Cells expressing IQGAP1 or a capping deficient IQGAP1(CD), unable to bind filament ends, on a plasmid did not display significant changes to cell morphology or actin filament structure compared to wildtype cells. However, cells expressing IQGAP1(CD) displayed significantly slower wound closure compared to cells with endogenous IQGAP1. These results suggest that IQGAP1-mediated capping is a physiologically relevant mechanism of regulating actin filament assembly. This study reveals a role for IQGAP1 as a transient capper that promotes protein exchange on filament ends, which may have implications in the regulation of actin filament lengths in cells.NAUpstate Medical UniversityBiochemistry & Molecular BiologyPh
Characterizing actin assembly mechanisms in ALS pathology
No full textAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to >sixty genes. However, the mechanistic details of onset are unknown and there is no known cure or effective treatment. Disease mechanisms linked to the dysfunction of the neuronal cytoskeleton are arguably the least explored, despite being involved in important cell processes. Eight cytoskeleton associated proteins are genetically linked to ALS onset, including tubulin-4A, spastin, kinesin-5A, dynactin subunit-1, neurofilament, peripherin, alsin, and profilin-1 (PFN1). Most of these genes are linked to microtubule dynamics, yet only PFN1 directly regulates actin assembly. Profilin is essential for many neuronal cell processes mediating the dynamics of lipids, nuclear signals, and the cytoskeleton in neurons. We performed a quantitative biochemical comparison of the impact of the eight ALS-associated profilin variants on actin assembly using single-molecule microscopy assays. The binding affinity of each ALS-related profilin for actin monomers was loosely correlated with the actin filament nucleation strength. The A20T, R136W, Q139L, and C71G variants failed to activate formin-based actin assembly, mostly explained by deficiencies in binding to poly-L-proline stretches in formin. In addition, chemical denaturation experiments suggest that the folding stability of some ALS profilin proteins impacts on actin assembly. Finally, to better understand the connection between the cytoskeleton and ALS we investigated the role of profilin and its direct binding ligand TDP-43 (TAR DNA-binding protein 43) on actin assembly. TDP-43 misregulation is a hallmark of nearly all forms of neurodegeneration, including an estimated 40% of familial ALS. Using advanced microscopy assays, we visualized purified TDP-43 forming biomolecular condensates. Actin was sequestered within TDP-43 condensates tempering total filament assembly. These observations were further exacerbated in the presence of profilin. These results indicate disruptions to actin assembly contribute to ALS and suggest therapeutic interventions targeting actin assembly may be a useful in treating ALS.NAUpstate Medical UniversityBiochemistry & Molecular BiologyMaster of Scienc
Arabidopsis capping protein senses cellular phosphatidic acid levels and transduces these into changes in actin cytoskeleton dynamics.
No full textAddendum to: J Li, JL Henty-Ridilla, S Huang, X Wang, L Blanchoin, CJ Staiger. Capping protein modulates the dynamic behavior of actin filaments in response to phosphatidic acid in Arabidopsis.. Plant Cell 2012; 24:International audiencePlants respond rapidly and precisely to a broad spectrum of developmental, biotic and abiotic cues. In many instances, signaling cascades involved in transducing this information result in changes to the cellular architecture and cytoskeletal rearrangements. Based originally on paradigms for animal cell signaling, phospholipids have received increased scrutiny as key intermediates for transmitting information to the actin cytoskeleton. Significantly, a wealth of biochemical data for plant actin-binding proteins (ABPs) demonstrates that many of these interact with phosphoinositide lipids in vitro. Moreover, phosphatidic acid (PA) has been identified not only as an abundant structural lipid in plants, but also as an intermediary in developmental and stress signaling pathways that lead to altered actin organization. Several years ago, the heterodimeric capping protein (CP) from Arabidopsis was demonstrated to bind PA and is negatively regulated by this lipid in vitro. Whether this form of regulation occurs in cells, however, remained a mystery. A new study, that combines live-cell imaging of cytoskeletal dynamics with reverse-genetic analyses in Arabidopsis, provides compelling new evidence that CP is inhibited from binding filament ends in the presence of PA in vivo. This allows rapid actin polymerization and increases in filament abundance following stimulation and could be one key factor in the physiological responses of plant cells to environmental stimuli
Global Resource Distribution: Allocation of Actin Building Blocks by Profilin
No full textHow cells regulate the distribution of a limited pool of actin between two competing structures has long been a mystery. Complementary studies from Suarez et al. (2015) and Rotty et al. (2015) now show that profilin controls the partitioning of actin monomers between competing actin networks assembled by Arp2/3 complex and formins or Ena/VASP
Sequential Derivatization of Polar Organic Compounds in Cloud Water Using O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine Hydrochloride, N, O-Bis(trimethylsilyl)trifluoroacetamide, and Gas-Chromatography/Mass Spectrometry Analysis
No full textCloud water samples from Whiteface Mountain, NY were used to develop a combined sampling and gas chromatography-mass spectrometric (GCMS) protocol for evaluating the complex mixture of highly polar organic compounds (HPOC) present in this atmospheric medium. Specific HPOC of interest were mono- and di keto-acids which are thought to originate from photochemical reactions of volatile unsaturated hydrocarbons from biogenic and manmade emissions and be a major fraction of atmospheric carbon. To measure HPOC mixtures and the individual keto-acids in cloud water, samples first must be derivatized for clean elution and measurement, and second, have low overall background of the target species as validated by GCMS analysis of field and laboratory blanks. Here, we discuss a dual derivatization method with PFBHA and BSTFA which targets only organic compounds that contain functional groups reacting with both reagents. The method also reduced potential contamination by minimizing the amount of sample processing from the field through the GCMS analysis steps. Once derivatized only gas chromatographic separation and selected ion monitoring (SIM) are needed to identify and quantify the polar organic compounds of interest. Concentrations of the detected total keto-acids in individual cloud water samples ranged from 27.8 to 329.3 ng mL-1 (ppb). Method detection limits for the individual HPOC ranged from 0.17 to 4.99 ng mL-1 and the quantification limits for the compounds ranged from 0.57 to 16.64 ng mL-1. The keto-acids were compared to the total organic carbon (TOC) results for the cloud water samples with concentrations of 0.607 to 3.350 mg L-1 (ppm). GCMS analysis of all samples and blanks indicated good control of the entire collection and analysis steps. Selected ion monitoring by GCMS of target keto-acids was essential for screening the complex organic carbon mixtures present at low ppb levels in cloud water. It was critical for ensuring high levels of quality assurance and quality control and for the correct identification and quantification of key marker compounds.Corrected proof of accepted manuscrip
Spawning ground surveys 2006-2007
No full textMatthew W. Hutchinson, Jessica L. Vogt and Alex Neerman.This archived document is maintained by the State Library of Oregon as part of the Oregon Documents Depository Program. It is for informational purposes and may not be suitable for legal purposes.Includes bibliographical references (pages 14-15).Mode of access: Internet from the Oregon Government Publications Collection.Text in English
Moravia E L\u27esperienza Autobiografica : Una Chiave Essenzial Per Decodificare I Soui Romanzi
No full textABSTRACT
MORAVIA E L\u27ESPERIENZA AUTOBIOGRAFICA : UNA CHIAVE ESSENZIALE PER DECODIFICARE I SOUI ROMANZI
By
JESSICA TOBY
May 2010
Advisor: Dr. Elena Past
Major: Italian
Degree: Master of Arts
This thesis takes a critical look at the physical and psychological experiences of Alberto Moravia as an adolescent and the integral role that they play in the development of his main literary characters. The key problem areas reside in decoding primary texts of the author alluding to psychological trauma in his childhood and analyzing the main protagonists of specific novels to reveal similarities between them and Moravia himself. The methods used in writing this thesis included examining primary texts, biographies, and theoretical works from a psycho-analytical perspective. In conclusion to this work, I have demonstrated that it is the responsibility of the critical reader to investigate an author\u27s autobiography in order to reveal hidden meaning in the text and unmask the true identity of his main protagonists
The availability of filament ends modulates actin stochastic dynamics in live plant cells.
No full textInternational audienceA network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament-filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls
Nine years of video landers at the Oregon Department of Fish & Wildlife's Marine Resources Program
No full textLeif K. Rasmuson, Kelly A. Lawrence, Gregory K. Krutzikowsky, Jessica L. Watson, Lindsay Aylesworth, Robert W. Hannah, Brett T. Rodomsky, Brittany Huntington, Keith Matteson, Ryan R. Easton.Title from PDF title page (viewed on April 1, 2022).This archived document is maintained by the State Library of Oregon as part of the Oregon Documents Depository Program. It is for informational purposes and may not be suitable for legal purposes.Includes bibliographical references (pages 40-46).Mode of access: Internet from the Oregon Government Publications Collection.Text in English
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