The Drosophila syncytial embryo is a powerful developmental model system for studying dynamic coordinated cytoskeletal rearrangements. Confocal microscopy has begun to reveal more about the cytoskeletal changes that occur during embryogenesis. Total internal reflection fluorescence (TIRF) microscopy provides a promising new approach for the visualization of cortical events with heightened axial resolution. We have applied TIRF microscopy to the Drosophila embryo to visualize cortical microtubule and actin dynamics in the syncytial blastoderm. Here, we describe the details of this technique, and report qualitative assessments of cortical microtubules and actin in the Drosophila syncytial embryo. In addition, we identified a peak of cortical microtubules during anaphase of each nuclear cycle in the syncytial blastoderm, and using images generated by TIRF microscopy, we quantitatively analyzed microtubule dynamics during this time.
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http://dx.doi.org/10.1002/dvdy.22076 | DOI Listing |
Chemphyschem
January 2025
Universität des Saarlandes, Biophysikalische Chemie FR 8.1 Chemie, Campus B 2 2, 66123, Saarbrücken, GERMANY.
The reaction of terrylene in p-terphenyl with molecular oxygen is reinvestigated by TIRF-microscopy with λexc = 488 nm or λexc = 561 nm and 488 nm. A similar range of fluorescent products is obtained under both experimental conditions with a reaction quantum yield Φr > 10-7 for those molecules which undergo the photoreaction. The majority of these oxygen-susceptible molecules reacts via an electronically relaxed, dark intermediate, presumably an endoperoxide, with a lifetime of
Front Immunol
January 2025
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.
Introduction: T cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response.
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December 2024
Graduate School of Life Sciences, Tohoku University, Miyagi, Japan.
The motile parameters of kinesin superfamily proteins are fundamental to intracellular transport. Single-molecule motility assays using total internal reflection fluorescence (TIRF) microscopy are a gold standard technique for measuring the motile parameters of kinesin motors. With this technique, one can evaluate the velocity, run length, and binding frequency of kinesins on microtubules by directly observing their motility.
View Article and Find Full Text PDFbioRxiv
December 2024
Department of Biochemistry, Brandeis University, Waltham, MA 02453.
Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter.
View Article and Find Full Text PDFbioRxiv
December 2024
Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois Chicago, Chicago, IL 60607, USA.
Tetraspanin proteins are closely associated with high-curvature membrane structures and play key roles in organizing membrane domains and regulating membrane signaling in immune cells. However, their specific roles in regulating T cell membrane signaling, particularly within the microvilli often characteristic of these cells, remain poorly understood. Here, we used Jurkat T cells as a model system and investigated CD81 as a member of the tetraspanin family.
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