Nuclear envelope formation during late mitosis includes rapid reassembly of several thousand complete nuclear pore complexes (NPCs). We observed the NPC reassembly process in cells undergoing division using a combination of genome edited reagents, and new imaging tools. We utilized lattice light-sheet microscopy and focused ion beam scanning electron microscopy to enable rapid, high-resolution, high-sensitivity 3D visualization of whole cells. Using this combination of reagents and imaging tools, which amounts to in vivo, single-molecule biochemistry, we discovered octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These remnants appeared as fenestrations and were associated with the mitotic ER and persisted through several cell divisions. We postulate that a yet-to-be-identified modification marks and “immortalizes” one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.
Next, I will introduce our next-generation microscope design – dubbed MOSAIC (multimodal optical scope with adaptive imaging correction), this is an ongoing unpublished project which combines several different modes of imaging with integrated light paths. In essence, this new microscope is designed to seamlessly switch between modes of imaging in order to alleviate the tradeoffs related to resolution, speed, invasiveness and imaging depth, which precludes any single optical microscopy to function optimally for a diverse set of biological specimens.
Inherited nuclear pore substructures template post-mitotic pore assembly: Article (Dev. Cell, 2021: www.sciencedirect.com/science/article/pii/S1534580721004445)
Expansion + Lattice Light Sheet Microscopy: Article (Science, 2019: www.science.org/doi/10.1126/science.aau8302)
Adaptive Optics + Lattice Light Sheet Microscopy Article (Science, 2018: www.science.org/doi/10.1126/science.aaq1392)
SPEAKER BIOGRAPHY
Srigokul (Gokul) Upadhyayula’s research interests bridge applied engineering with basic science. He studied the charge transfer properties of cyanine dyes and bioinspired electrets using ultra-fast femtosecond spectroscopy as a doctoral student with Prof. Valentine Vullev at University of California, Riverside. Gokul joined Tom Kirchhausen’s group at Harvard Medical School / Boston Children’s Hospital as a postdoctoral fellow, where he focused on questions addressed at a molecular level using lattice light-sheet microscopy (LLSM) with high temporal and spatial resolution. In parallel, Gokul joined Eric Betzig’s group at Janelia Research Campus as a visiting scientist, where he collaborated on the adaptive optical LLSM project to investigate sub-cellular dynamics within the natural environment of multicellular organisms such as zebrafish embryos, and on the expansion microscopy + LLSM project to image the entire fly brain and mouse cortical column with synaptic resolution. Gokul became an Assistant Professor in the Department of Pediatrics at Harvard Medical School since 2018. In 2019, Gokul moved to join UC Berkeley’s faculty and built the Advanced BioImaging Center (ABC) as its scientific director.
Gokul, along with ABC co-founders Nobel Laureate Eric Betzig, Xavier Darzacq, Doug Koshland, and Robert Tjian, aims to bring scientists with broad specialties (instrumentation, biology, applied mathematics, and computer science) together and provide free access to advanced imaging systems and resources. To start, Gokul and his team built two cutting-edge adaptive optical multi-functional microscopes to enable imaging across scales spanning several orders of magnitude in space and time, with, for example, specimens up to several millimeters in size, or over imaging sessions lasting up to multiple days. Consequently, the greatest challenge the users face is the ability to visualize, analyze and understand the explosively large quantities of immensely complex data. The primary goal of the ABC is therefore to provide both cutting-edge microscopy, and dedicated human and hardware resources capable of handling tera- to petabyte scale projects and developing robust, open source computational workflows that allow scientists to extract biologically meaningful insights.
