@article {69756, title = {Periodic ER-plasma membrane junctions support long-range Ca signal integration in dendrites.}, journal = {Cell}, volume = {188}, year = {2025}, month = {01/2025}, pages = {484-500.e22}, abstract = {

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~{}1\ μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20\ μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

}, keywords = {Animals, Calcium, Calcium Signaling, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cell Membrane, Dendrites, Endoplasmic Reticulum, Hippocampus, Humans, Male, Mice, Rats, Ryanodine Receptor Calcium Release Channel}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.11.029}, author = {Benedetti, Lorena and Fan, Ruolin and Weigel, Aubrey V and Moore, Andrew S and Houlihan, Patrick R and Kittisopikul, Mark and Park, Grace and Petruncio, Alyson and Hubbard, Philip M and Pang, Song and Xu, C Shan and Hess, Harald F and Saalfeld, Stephan and Rangaraju, Vidhya and Clapham, David E and De Camilli, Pietro and Ryan, Timothy A and Lippincott-Schwartz, Jennifer} } @article {69704, title = {Periodic ER-plasma membrane junctions support long-range Ca signal integration in dendrites.}, journal = {Cell}, year = {2024}, month = {2024 Dec 18}, abstract = {

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at \~{}1\ μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca channels and ER Ca-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca homeostasis, and local activation of the Ca/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca release at ER-PM junctions over 20\ μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2024.11.029}, author = {Benedetti, Lorena and Fan, Ruolin and Weigel, Aubrey V and Moore, Andrew S and Houlihan, Patrick R and Kittisopikul, Mark and Park, Grace and Petruncio, Alyson and Hubbard, Philip M and Pang, Song and Xu, C Shan and Hess, Harald F and Saalfeld, Stephan and Rangaraju, Vidhya and Clapham, David E and De Camilli, Pietro and Ryan, Timothy A and Lippincott-Schwartz, Jennifer} } @article {67178, title = {Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites}, journal = {Current Protocols}, volume = {1}, year = {2021}, month = {2021 Mar 03}, abstract = {

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein{\textquoteright}s intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. {\textcopyright} 2021 Wiley Periodicals LLC.

}, issn = {2691-1299}, doi = {https://doi.org/10.1002/cpz1.71}, url = {https://doi.org/10.1002/cpz1.71}, author = {Benedetti, Lorena} } @article {67646, title = {YAP1 nuclear efflux and transcriptional reprograming follow membrane diminution upon VSV-G-induced cell fusion.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {2021 Jul 23}, pages = {4502}, abstract = {

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells.\ RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-24708-2}, author = {Feliciano, Daniel and Ott, Carolyn M and Isabel Espinosa Medina and Weigel, Aubrey V and Benedetti, Lorena and Milano, Kristin M and Tang, Zhonghua and Lee, Tzumin and Kliman, Harvey J and Guller, Seth M and Lippincott-Schwartz, Jennifer} } @article {65262, title = {Nanoscale subcellular architecture revealed by multicolor three-dimensional salvaged fluorescence imaging.}, journal = {Nature Methods}, volume = {17}, year = {2020}, month = {2020 Jan 06}, pages = {225-231}, abstract = {

Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using {\textquoteright}salvaged fluorescence{\textquoteright}. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

}, issn = {1548-7105}, doi = {10.1038/s41592-019-0676-4}, author = {Zhang, Yongdeng and Schroeder, Lena K and Lessard, Mark D and Kidd, Phylicia and Chung, Jeeyun and Song, Yuanbin and Benedetti, Lorena and Li, Yiming and Ries, Jonas and Grimm, Jonathan B and Lavis, Luke D and De Camilli, Pietro and Rothman, James E and Baddeley, David and Bewersdorf, Joerg} } @article {65827, title = {Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Nov 11}, abstract = {

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these {\textquoteright}enhanced{\textquoteright} Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

}, issn = {2050-084X}, doi = {10.7554/eLife.63230}, author = {Benedetti, Lorena and Marvin, Jonathan S and Falahati, Hanieh and Guill{\'e}n-Samander, Andres and Looger, Loren L and De Camilli, Pietro} }