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Elodie Sartorel, Caner Ünlü, Mini Jose, Aurélie Massoni-Laporte, Julien Meca, Jean-Baptiste Sibarita, Derek McCusker
The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatio-temporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters...
April 18, 2018: Molecular Biology of the Cell
Adekunle T Bademosi, Elsa Lauwers, Rumelo Amor, Patrik Verstreken, Bruno van Swinderen, Frédéric A Meunier
An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane...
January 14, 2018: Journal of Visualized Experiments: JoVE
Anthony Fernandez, Markville Bautista, Ramunas Stanciauskas, Taerin Chung, Fabien Pinaud
Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells...
August 9, 2017: ACS Applied Materials & Interfaces
Mikael Garcia, Cécile Leduc, Matthieu Lagardère, Amélie Argento, Jean-Baptiste Sibarita, Olivier Thoumine
Neuronal growth cones move forward by dynamically connecting actin-based motility to substrate adhesion, but the mechanisms at the individual molecular level remain unclear. We cultured primary neurons on N-cadherin-coated micropatterned substrates, and imaged adhesion and cytoskeletal proteins at the ventral surface of growth cones using single particle tracking combined to photoactivated localization microscopy (sptPALM). We demonstrate transient interactions in the second time scale between flowing actin filaments and immobilized N-cadherin/catenin complexes, translating into a local reduction of the actin retrograde flow...
June 2, 2015: Proceedings of the National Academy of Sciences of the United States of America
Amulya N Shrivastava, Pamela C Rodriguez, Antoine Triller, Marianne Renner
Adenosine triphosphate (ATP)-gated P2X7 receptors (P2X7Rs) are members of the purinergic receptor family that are expressed in several cell types including neurons. A high concentration of ATP is required for the channel opening of P2X7Rs compared to other members of this receptor family. Recent work suggests that ATP binding to members of the P2X receptor family determines the diffusion and localization of these receptors on the plasma membrane of neurons. Here, we employed single particle tracking photoactivated localization microscopy (sptPALM) to study the diffusion and ATP-dependence of rat P2X7Rs...
2013: Frontiers in Cellular Neuroscience
Suliana Manley, Jennifer M Gillette, Jennifer Lippincott-Schwartz
Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues...
2010: Methods in Enzymology
Fedor V Subach, George H Patterson, Malte Renz, Jennifer Lippincott-Schwartz, Vladislav V Verkhusha
Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes...
May 12, 2010: Journal of the American Chemical Society
Suliana Manley, Jennifer M Gillette, George H Patterson, Hari Shroff, Harald F Hess, Eric Betzig, Jennifer Lippincott-Schwartz
We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.
February 2008: Nature Methods
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