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89 Publications

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    06/10/24 | Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM
    Renganathan B, Moore AS, Yeo W, Petruncio A, Ackerman D, Wiegel A, CellMap Team , Pasolli HA, Xu CS, Hess HF, Serpinskaya AS, Zhang HF, Lippincott-Schwartz J, Gelfand VI
    bioRxiv. 2024 Jun 10:. doi: 10.1101/2024.06.10.598346

    Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

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    05/31/24 | Periodic ER-plasma membrane junctions support long-range Ca2+ signal integration in dendrites
    Benedetti L, Fan R, Weigel AV, Moore AS, Houlihan PR, Kittisopikul M, Park G, Petruncio A, Hubbard PM, Pang S, Xu CS, Hess HF, Saalfeld S, Rangaraju V, Clapham DE, De Camilli P, Ryan TA, Lippincott-Schwartz J
    bioRxiv. 2024 May 31:. doi: 10.1101/2024.05.27.596121

    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 Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery facilitating voltage-independent signal transmission and ryanodine receptor-dependent Ca2+ release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ 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.HighlightsPeriodic ER-PM junctions tile neuronal dendritic plasma membrane in rodent and fly.ER-PM junctions are populated by ER tethering and Ca2+ release and influx machinery.ER-PM junctions act as sites for local activation of CaMKII.Local spine activation drives Ca2+ release from RyRs at ER-PM junctions over 20 μm.

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    06/04/24 | The physical and cellular mechanism of structural color change in zebrafish.
    Gur D, Moore AS, Deis R, Song P, Wu X, Pinkas I, Deo C, Iyer N, Hess HF, Hammer JA, Lippincott-Schwartz J
    Proc Natl Acad Sci U S A. 2024 Jun 04;121(23):e2308531121. doi: 10.1073/pnas.2308531121

    Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

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    05/13/24 | Ultrastructural differences impact cilia shape and external exposure across cell classes in the visual cortex
    Ott CM, Torres R, Kuan T, Kuan A, Buchanan J, Elabbady L, Seshamani S, Bodor AL, Collman F, Bock DD, Lee WC, da Costa NM, Lippincott-Schwartz J
    Curr Biol. 2024 May 13:. doi: 10.1016/j.cub.2024.04.043

    A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.

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    05/07/24 | YAP condensates are highly organized hubs
    Siyuan Hao , Ye Jin Lee , Nadav Benhamou Goldfajn , Eduardo Flores , Jindayi Liang , Hannah Fuehrer , Justin Demmerle , Jennifer Lippincott-Schwartz , Zhe Liu , Shahar Sukenik , Danfeng Cai
    iScience. 2024 May 07:109927. doi: https://doi.org/10.1016/j.isci.2024.109927

    YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

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    04/06/24 | COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.
    Liao Y, Pang S, Li W, Shtengel G, Choi H, Schaefer K, Xu CS, Lippincott-Schwartz J
    Dev Cell. 2024 Apr 06:. doi: 10.1016/j.devcel.2024.03.027

    Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.

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    02/22/24 | CSPP1 stabilizes microtubules by capping both plus and minus ends.
    Wang Z, Wang W, Liu S, Yang F, Liu X, Hua S, Zhu L, Xu A, Hill DL, Wang D, Jiang K, Lippincott-Schwartz J, Liu X, Yao X
    Journal of Molecular Cell Biology. 2024 Feb 22:. doi: 10.1093/jmcb/mjae007

    Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

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    02/21/24 | Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.
    Dou Z, Liu R, Gui P, Fu C, Lippincott-Schwartz J, Yao X, Liu X
    Molecular Biology of the Cell. 2024 Feb 21:mbcE23090379. doi: 10.1091/mbc.E23-09-0379

    Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

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    05/04/24 | Host ZCCHC3 blocks HIV-1 infection and production by a dual mechanism
    Binbin Yi , Yuri L Tanaka , Hidetaka Kosako , Erika P Butlertanaka , Prabuddha Sengupta , Jennifer Lippincott-Schwartz , Akatsuki Saito , Shige H. Yoshimura
    iScience. 05/2024:. doi: 10.1101/2023.06.14.544911

    Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. While anti-human immunodeficiency virus type 1 (HIV-1) host restriction factors have been identified, most of them are antagonized by viral proteins. This has severely hindered their development in anti-HIV-1 therapy. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel anti-HIV-1 factor that is not antagonized by viral proteins. ZCCHC3 suppresses production of HIV-1 and other retroviruses. We show that ZCCHC3 acts by binding to Gag nucleocapsid protein via zinc-finger motifs. This prevents interaction between the Gag nucleocapsid protein and viral genome and results in production of genome-deficient virions. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. Such a dual antiviral mechanism is distinct from that of any other known host restriction factors. Therefore, ZCCHC3 is a novel potential target in anti-HIV-1 therapy.

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    01/24/24 | Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.
    Obara CJ, Nixon-Abell J, Moore AS, Riccio F, Hoffman DP, Shtengel G, Xu CS, Schaefer K, Pasolli HA, Masson J, Hess HF, Calderon CP, Blackstone C, Lippincott-Schwartz J
    Nature. 2024 Jan 24;626(7997):169-176. doi: 10.1038/s41586-023-06956-y

    To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

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