Biology subjects

Lippincott-Schwartz, J.

Publications and source records attributed to Lippincott-Schwartz, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Host ZCCHC3 blocks HIV-1 infection and production by a dual mechanism

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.

microbiology↗

bioRxiv2023

Simultaneous photoactivation and high-speed structural tracking reveal diffusion-dominated motion in the endoplasmic reticulum

The endoplasmic reticulum (ER) is a structurally complex, membrane-enclosed compartment that stretches from the nuclear envelope to the extreme periphery of eukaryotic cells. The organelle is crucial for numerous distinct cellular processes, but how these processes are spatially regulated within the structure is unclear. Traditional imaging-based approaches to understanding protein dynamics within the organelle are limited by the convoluted structure and rapid movement of molecular components. Here, we introduce a combinatorial imaging and machine learning-assisted image analysis approach to track the motion of photoactivated proteins within the ER of live cells. We find that simultaneous knowledge of the underlying ER structure is required to accurately analyze fluorescently-tagged protein redistribution, and after appropriate structural calibration we see all proteins assayed show signatures of Brownian diffusion-dominated motion over micron spatial scales. Remarkably, we find that in some cells the ER structure can be explored in a highly asymmetric manner, likely as a result of uneven connectivity within the organelle. This remains true independently of the size or folding state of the fluorescently-tagged molecules, suggesting a potential role for ER connectivity in driving spatially regulated biology in eukaryotes.

cell biology↗

bioRxiv2023

Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome

Cristae are high curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous mechanisms for lipids have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the IMM against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. The model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that CL is essential in low oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of CL is dependent on the surrounding lipid and protein components of the IMM. SynopsisO_LIcritical lipidic breakpoint for yeast mitochondria phenocopies the loss of cristae-shaping proteins in the IMM. C_LIO_LIsaturation controls membrane mechanical properties and modulates ATP synthase oligomerization. C_LIO_LImitochondrial-specific lipid cardiolipin can functionally compensate for increased phospholipid saturation and is required for cristae formation in low oxygen environments. C_LIO_LImathematical model for cristae membrane tubules predicts a snapthrough instability mediated by both protein and lipid-encoded curvatures. C_LI Synopsis Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/532310v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@196aefforg.highwire.dtl.DTLVardef@1bafcadorg.highwire.dtl.DTLVardef@122f502org.highwire.dtl.DTLVardef@35afd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

YAP condensates are highly organized hubs for YAP/TEAD transcription

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. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule 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.

cell biology↗

bioRxiv2022

Motion of single molecular tethers reveals dynamic subdomains at ER-mitochondria contact sites

To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites3. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle4,5. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation6,7, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

cell biology↗

bioRxiv2021

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form1. This transformation is facilitated by the engulfment and degradation of excess axonal branches and inappropriate synapses by surrounding glial cells, including microglia and astrocytes2,3. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia has made it difficult to determine the contribution of these and other glial cell types to this process. Here, we used large scale, serial electron microscopy (ssEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors4, frequently surrounded terminal axon branches and included numerous phagolysosomes (PLs) containing fragments of axons and presynaptic terminals. Single- nucleus RNA sequencing indicated that cortical OPCs express key phagocytic genes, as well as neuronal transcripts, consistent with active axonal engulfment. PLs were ten times more abundant in OPCs than in microglia in P36 mice, and declined with age and lineage progression, suggesting that OPCs contribute very substantially to refinement of neuronal circuits during later phases of cortical development.

neuroscience↗

bioRxiv2021

Vacuole phase-partitioning boosts mitochondria activity and cell lifespan through an inter-organelle lipid pipeline

Functional linkage between mitochondria and lysosomes is crucial for survival under starvation and lifespan extension. Despite such co-dependency, the supportive pathways connecting mitochondria and lysosomes remain unclear. Here, we identify an inter-organelle lipid trafficking pathway linking yeast vacuole and mitochondria that results in increased mitochondria growth and respiratory activity under glucose starvation. The pathway depends on vacuolar phase-separated, lipid domains, which provide zones for: activation of the vacuolar proton pump; lipid droplet (LD) docking and internalization; and, lipid transfer from vacuole-to-ER-to-mitochondria. Partitioned vacuolar domains form through a specialized type of macro-autophagy, triggered only under acute glucose starvation, that delivers sterol-rich, endosomal-derived lipids to the vacuole. To balance this lipid influx, the vacuole reroutes lipids back to the ER to support both LD biogenesis and mitochondria growth and activity. Energy produced by enhanced mitochondrial activity then feeds back to support the inter-organelle lipid trafficking pathways to ensure survival under nutrient stress.

cell biology↗

bioRxiv2021

Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrites satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.

neuroscience↗

bioRxiv2020

Automatic whole cell organelle segmentation in volumetric electron microscopy

Cells contain hundreds of different organelle and macromolecular assemblies intricately organized relative to each other to meet any cellular demands. Obtaining a complete understanding of their organization is challenging and requires nanometer-level, threedimensional reconstruction of whole cells. Even then, the immense size of datasets and large number of structures to be characterized requires generalizable, automatic methods. To meet this challenge, we developed an analysis pipeline for comprehensively reconstructing and analyzing the cellular organelles in entire cells imaged by focused ion beam scanning electron microscopy (FIB-SEM) at a near-isotropic size of 4 or 8 nm per voxel. The pipeline involved deep learning architectures trained on diverse samples for automatic reconstruction of 35 different cellular organelle classes - ranging from endoplasmic reticulum to microtubules to ribosomes - from multiple cell types. Automatic reconstructions were used to directly quantify various previously inaccessible metrics about these structures, including their spatial interactions. We show that automatic organelle reconstructions can also be used to automatically register light and electron microscopy images for correlative studies. We created an open data and open source web repository, OpenOrganelle, to share the data, computer code, and trained models, enabling scientists everywhere to query and further reconstruct the datasets.

cell biology↗

bioRxiv2020

Deuteration improves small-molecule fluorophores

Fluorescence microscopy relies on dyes that absorb short-wavelength photons and emit longer-wavelength light. In addition to this fluorescence process, dyes can undergo other photochemical reactions that result in spectral shifts and irreversible photobleaching. Increases in brightness, chromostability, and photostability of fluorescent dyes are therefore crucial for advancing the frontier of bioimaging. Here, we describe a general approach to improve small-molecule fluorophores using deuteration. Incorporating deuterium into the alkylamino substituents of rhodamines and other dyes improves fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching. These compounds are easily synthesized and show improved performance in cellular imaging experiments.

cell biology↗

bioRxiv2020

Image-based pooled whole genome CRISPR screening for Parkin and TFEB subcellular localization

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

Label-free single-instance protein detection in vitrified cells

A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of images-- calculated on the basis of known structural data--as search templates to detect targets as small as the "head" domain (350 kDa) of the ribosomes small subunit in single-tilt electron cryo-micrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryo-electron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling.

biophysics↗

bioRxiv2020

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

Skin color patterns are ubiquitous in nature, evolve rapidly, and impact social behavior1, predator avoidance2, and protection from ultraviolet irradiation3. A leading model system for vertebrate skin patterning is the zebrafish4-7; its alternating blue stripes and yellow interstripes depend on guanine crystal-containing cells called iridophores that reflect light. It was suggested that the zebrafishs alternating color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes7-9. When we tracked iridophores, however, we found they did not migrate between stripes and interstripes but instead differentiated and proliferated in place based on their micro-environment. RNA seq analysis further revealed stripe and interstripe iridophores had different transcriptomic states, while cryogenic scanning electron microscopy and micro-X-ray diffraction showed they had different guanine crystal organizations and responsiveness to norepinephrine, all indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present a new model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, subcellular organelles arise in stripe and interstripe zones by in situ differentiation. In this model, pattern phenotype depends not only on interactions among pigment cells that affect their arrangements, but also on factors that specify subcellular organization and physiological responsiveness of specialized organelles.

developmental biology↗

bioRxiv2020

Nicotine exposure and neuronal activity regulate Golgi membrane dispersal and distribution

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of 4{beta}2-type nicotinic acetylcholine receptors (4{beta}2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required 4{beta}2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotines addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger "ministacks", similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of 4{beta}2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

neuroscience↗

bioRxiv2019

Optimization and functionalization of red-shifted rhodamine dyes

Expanding the palette of fluorescent dyes is vital for pushing the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore due to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups.

cell biology↗

bioRxiv2019

Transcriptional reprogramming in fused cells is triggered by plasma-membrane diminution

Developing cells divide and differentiate, and in many tissues, such as bone, muscle, and placenta, cells fuse acquiring specialized functions. While it is known that fused-cells are differentiated, it is unclear what mechanisms trigger the programmatic-change, and whether cell-fusion alone drives differentiation. To address this, we employed a fusogen-mediated cell-fusion system involving undifferentiated cells in tissue culture. RNA-seq analysis revealed cell-fusion initiates a dramatic transcriptional change towards differentiation. Dissecting the mechanisms causing this reprogramming, we observed that after cell-fusion plasma-membrane surface area decreases through increased endocytosis. Consequently, glucose-transporters are internalized, and cytoplasmic-glucose and ATP transiently decrease. This low-energetic state activates AMPK, which inhibits YAP1, causing cell-cycle arrest. Impairing either endocytosis or AMPK prevents YAP1 inhibition and cell-cycle arrest after fusion. Together these data suggest that cell-fusion-induced differentiation does not need to rely on extrinsic-cues; rather the plasma-membrane diminishment forced by the geometric-transformations of cell-fusion cause transient cell-starvation that induces differentiation.

cell biology↗

bioRxiv2019

Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells

Living cells function through the spatial compartmentalization of thousands of distinct proteins serving a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) has emerged as a pathway to directly view nanoscale protein relationships to the underlying global ultrastructure, but has traditionally suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional correlative cryogenic SR and focused ion beam milled block-face EM across entire vitreously frozen cells that addresses these issues by preserving native ultrastructure and enabling independent SR and EM workflow optimization. Application to a variety of biological systems revealed a number of unexpected protein-ultrastructure relationships and underscored the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

cell biology↗

bioRxiv2019

ER membranes exhibit phase behavior at sites of organelle contact

The plasma membrane of cells exhibits phase behavior that allows transient concentration of specific proteins and lipids, giving rise to functionally dynamic and diverse nanoscopic domains. This phase behavior is observable in giant plasma membrane-derived vesicles, in which microscopically visible, liquid-ordered (Lo) and liquid-disordered (Ld) lipid domains form upon a shift to low temperatures. The extent such phase behavior exists in the membrane of the endoplasmic reticulum (ER) of cells remains unclear. To explore the phase behavior of the ER membrane in cells, we used hypotonic cell swelling to generate Large Intra-Cellular Vesicles (LICVs) from the ER in cells. ER LICVs retained their lumenal protein content, could be retubulated into an ER network, and maintained stable inter-organelle contacts, where protein tethers are concentrated at these contacts. Notably, upon temperature reduction, ER LICVs underwent reversible phase separation into microscopically-visible Lo and Ld lipid domains. The Lo lipid domains marked ER contact sites with other organelles. These findings demonstrate that LICVs provide an important model system for studying the biophysical properties of intracellular organelles in cells.\n\nSignificance StatementPrior work has demonstrated that the plasma membrane can phase separate into microscopically visible Lo and Ld domains with distinct lipid and protein content. However, such behavior on the ER membrane has not been experimentally observed, even though the ER contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner at these contacts. We find here that hypotonic treatment generates Large Intra-Cellular Vesicles from the endoplasmic reticulum and other membrane-bound organelles in cells, enabling the study of phase behavior on the ER membrane. We show that ER membranes can be reversibly phase separated into microscopically-observable, Lo and Ld domains. ER LICVs also maintained stable inter-organelle contact sites in cells, with organelle tethers concentrated at these contacts.

biophysics↗