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2691 Janelia Publications

Showing 121-130 of 2691 results
12/24/24 | Days-old zebrafish rapidly learn to recognize threatening agents through noradrenergic and forebrain circuits.
Zocchi D, Nguyen M, Marquez-Legorreta E, Siwanowicz I, Singh C, Prober DA, Hillman EM, Ahrens MB
Curr Biol. 2024 Dec 19:. doi: 10.1016/j.cub.2024.11.057

Animals need to rapidly learn to recognize and avoid predators. This ability may be especially important for young animals due to their increased vulnerability. It is unknown whether, and how, nascent vertebrates are capable of such rapid learning. Here, we used a robotic predator-prey interaction assay to show that 1 week after fertilization-a developmental stage where they have approximately 1% the number of neurons of adults-zebrafish larvae rapidly and robustly learn to recognize a stationary object as a threat after the object pursues the fish for ∼1 min. Larvae continue to avoid the threatening object after it stops moving and can learn to distinguish threatening from non-threatening objects of a different color. Whole-brain functional imaging revealed the multi-timescale activity of noradrenergic neurons and forebrain circuits that encoded the threat. Chemogenetic ablation of those populations prevented the learning. Thus, a noradrenergic and forebrain multiregional network underlies the ability of young vertebrates to rapidly learn to recognize potential predators within their first week of life.

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12/31/24 | Discovery of neuronal cell types by pairing whole cell reconstructions with RNA expression profiles
The MouseLight Project Team , Ferreira TA, Eddison M, Copeland M, Lay M, Tenshaw E, Weldon M, Schauder D, Olbris DJ, Rokicki K, Spruston N, Tillberg PW, Korff W, Dudman JT
bioRxiv. 12/2024:. doi: 10.1101/2024.12.30.630829

Effective classification of neuronal cell types requires both molecular and morphological descriptors to be collected in situ at single cell resolution. However, current spatial transcriptomics techniques are not compatible with imaging workflows that successfully reconstruct the morphology of complete axonal projections. Here, we introduce a new methodology that combines tissue clearing, submicron whole-brain two photon imaging, and Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to assign molecular identities to fully reconstructed neurons in the mouse brain, which we call morphoFISH. We used morphoFISH to molecularly identify a previously unknown population of cingulate neurons projecting ipsilaterally to the dorsal striatum and contralaterally to higher-order thalamus. By pairing whole-brain morphometry, improved techniques for nucleic acid preservation and spatial gene expression, morphoFISH offers a quantitative solution for discovery of multimodal cell types and complements existing techniques for characterization of increasingly fine-grained cellular heterogeneity in brain circuits.Competing Interest StatementThe authors have declared no competing interest.

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12/24/24 | Pearling Drives Mitochondrial DNA Nucleoid Distribution
Landoni JC, Lycas MD, Macuada J, Jaccard R, Obara CJ, Moore AS, Ben Nejma S, Hoffman D, Lippincott-Schwartz J, Marshall W, Sturm G, Manley S
bioRxiv. 12/2024:. doi: 10.1101/2024.12.21.629917

The regular distribution of mitochondrial DNA-containing nucleoids is essential for mitochondrial function and genome inheritance; however, the underlying mechanisms remain unknown. Our data reveal that mitochondria frequently undergo spontaneous and reversible pearling - a biophysical instability in which tubules undulate into regularly spaced beads. We discovered that pearling imposes a characteristic length scale, simultaneously mediating nucleoid disaggregation and establishing inter-nucleoid distancing with near-maximally achievable precision. Cristae invaginations play a dual role: lamellar cristae density determines pearling frequency and duration, and preserves the resulting nucleoid spacing after recovery. The distribution of mitochondrial genomes is thus fundamentally governed by the interplay between spontaneous pearling and cristae ultrastructure.

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12/18/24 | Periodic ER-plasma membrane junctions support long-range Ca 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
Cell. 2024 Dec 18:. doi: 10.1016/j.cell.2024.11.029

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.

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12/20/24 | Permanent cilia loss during cerebellar granule cell neurogenesis involves withdrawal of cilia maintenance and centriole capping.
Constable S, Ott CM, Lemire AL, White K, Xun Y, Lim A, Lippincott-Schwartz J, Mukhopadhyay S
Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2408083121. doi: 10.1073/pnas.2408083121

Brain neurons utilize the primary cilium as a privileged compartment to detect and respond to extracellular ligands such as Sonic hedgehog (SHH). However, cilia in cerebellar granule cell (GC) neurons disassemble during differentiation through ultrastructurally unique intermediates, a process we refer to as cilia deconstruction. In addition, mature neurons do not reciliate despite having docked centrioles. Here, we identify molecular changes that accompany cilia deconstruction and centriole docking in GC neurons. We used single cell transcriptomic and immunocytological analyses to compare the transcript levels and subcellular localization of proteins between progenitor, differentiating, and mature GCs. Differentiating GCs lacked transcripts for key activators of premitotic cilia resorption, indicating that cilia disassembly in differentiating cells is distinct from premitotic cilia resorption. Instead, during differentiation, transcripts of many genes required for cilia maintenance-specifically those encoding components of intraflagellar transport, pericentrosomal material, and centriolar satellites-decreased. The abundance of several corresponding proteins in and around cilia and centrosomes also decreased. These changes coincided with downregulation of SHH signaling prior to differentiation, even in a mutant with excessive SHH activation. Finally, mother centrioles in maturing granule neurons recruited the cap complex protein, CEP97. These data suggest that a global, developmentally programmed decrease in cilium maintenance in differentiating GCs mediates cilia deconstruction, while capping of docked mother centrioles prevents cilia regrowth and dysregulated SHH signaling. Our study provides mechanistic insights expanding our understanding of permanent cilia loss in multiple tissue-specific contexts.

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01/01/25 | Structural dynamics and binding of Caenorhabditis elegans lifespan-extending lipid binding protein-3 to polyunsaturated fatty acids.
Cuevas AR, Tillman MC, Wang MC, Ortlund EA
Protein Sci. 2025 Jan 01;34(1):e5249. doi: 10.1002/pro.5249

Intracellular lipid binding proteins (iLBPs) play crucial roles in lipid transport and cellular metabolism across the animal kingdom. Recently, a fat-to-neuron axis was described in Caenorhabditis elegans, in which lysosomal activity in the fat liberates polyunsaturated fatty acids (PUFAs) that signal to neurons and extend lifespan with durable fecundity. In this study, we investigate the structure and binding mechanisms of a lifespan-extending lipid chaperone, lipid binding protein-3 (LBP-3), which shuttles dihomo-γ-linolenic (DGLA) acid from intestinal fat to neurons. We present the first high-resolution crystal structure of LBP-3, which reveals a classic iLBP fold with an unexpected and unique homodimeric arrangement via interstrand interactions that is incompatible with ligand binding. We identify key ionic interactions that mediate DGLA binding within the lipid binding pocket. Molecular dynamics simulations further elucidate LBP-3's preferential binding to DGLA due to its rotational freedom and access to favorable binding conformations compared to other 20-carbon PUFAs. We also propose that LBP-3 dimerization may be a unique regulatory mechanism for lipid chaperones.

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12/24/24 | Days-old zebrafish rapidly learn to recognize threatening agents through noradrenergic and forebrain circuits.
Zocchi D, Nguyen M, Marquez-Legorreta E, Siwanowicz I, Singh C, Prober DA, Hillman EM, Ahrens MB
Curr Biol. 12/2024;35(1):163-176.e4. doi: 10.1016/j.cub.2024.11.057

Animals need to rapidly learn to recognize and avoid predators. This ability may be especially important for young animals due to their increased vulnerability. It is unknown whether, and how, nascent vertebrates are capable of such rapid learning. Here, we used a robotic predator-prey interaction assay to show that 1 week after fertilization-a developmental stage where they have approximately 1% the number of neurons of adults-zebrafish larvae rapidly and robustly learn to recognize a stationary object as a threat after the object pursues the fish for ∼1 min. Larvae continue to avoid the threatening object after it stops moving and can learn to distinguish threatening from non-threatening objects of a different color. Whole-brain functional imaging revealed the multi-timescale activity of noradrenergic neurons and forebrain circuits that encoded the threat. Chemogenetic ablation of those populations prevented the learning. Thus, a noradrenergic and forebrain multiregional network underlies the ability of young vertebrates to rapidly learn to recognize potential predators within their first week of life.

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12/16/24 | Development of a First-in-Class RIPK1 Degrader to Enhance Antitumor Immunity
Xin Yu , Dong Lu , Xiaoli Qi , Hanfeng Lin , Bryan L. Holloman , Feng Jin , Longyong Xu , Lang Ding , Weiyi Peng , Meng C. Wang , Xi Chen , Jin Wang
Nat Commun. 2024 Dec 16;15(1):10683. doi: 10.1038/s41467-024-55006-2

The scaffolding function of receptor interacting protein kinase 1 (RIPK1) confers intrinsic and extrinsic resistance to immune checkpoint blockades (ICBs) and emerges as a promising target for improving cancer immunotherapies. To address the challenge posed by a poorly defined binding pocket within the intermediate domain of RIPK1, here we harness proteolysis targeting chimera (PROTAC) technology to develop a RIPK1 degrader, LD4172. LD4172 exhibits potent and selective RIPK1 degradation both in vitro and in vivo. Degradation of RIPK1 by LD4172 triggers immunogenic cell death, enhances tumor-infiltrating lymphocyte responses, and sensitizes tumors to anti-PD1 therapy in female C57BL/6J mice. This work reports a RIPK1 degrader that serves as a chemical probe for investigating the scaffolding functions of RIPK1 and as a potential therapeutic agent to enhance tumor responses to ICBs therapy.

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12/16/24 | Ketamine induces plasticity in a norepinephrine-astroglial circuit to promote behavioral perseverance.
Duque M, Chen AB, Hsu E, Narayan S, Rymbek A, Begum S, Saher G, Cohen AE, Olson DE, Li Y, Prober DA, Bergles DE, Fishman MC, Engert F, Ahrens MB
Neuron. 2024 Dec 16(113):1-15. doi: 10.1016/j.neuron.2024.11.011

Transient exposure to ketamine can trigger lasting changes in behavior and mood. We found that brief ketamine exposure causes long-term suppression of futility-induced passivity in larval zebrafish, reversing the "giving-up" response that normally occurs when swimming fails to cause forward movement. Whole-brain imaging revealed that ketamine hyperactivates the norepinephrine-astroglia circuit responsible for passivity. After ketamine washout, this circuit exhibits hyposensitivity to futility, leading to long-term increased perseverance. Pharmacological, chemogenetic, and optogenetic manipulations show that norepinephrine and astrocytes are necessary and sufficient for ketamine's long-term perseverance-enhancing aftereffects. In vivo calcium imaging revealed that astrocytes in adult mouse cortex are similarly activated during futility in the tail suspension test and that acute ketamine exposure also induces astrocyte hyperactivation. The cross-species conservation of ketamine's modulation of noradrenergic-astroglial circuits and evidence that plasticity in this pathway can alter the behavioral response to futility hold promise for identifying new strategies to treat affective disorders.

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12/11/24 | TUDCA modulates drug bioavailability to regulate resistance to acute ER stress in Saccharomyces cerevisiae
Chadwick SR, Stack-Couture S, Berg MD, Gregorio SD, Lung B, Genereaux J, Moir RD, Brandl CJ, Willis IM, Snapp EL, Lajoie P
Mol Biol Cell. 2024 Dec 11:mbcE24040147. doi: 10.1091/mbc.E24-04-0147

Cells counter accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) through activation of the Unfolded Protein Response (UPR). Small molecules termed chemical chaperones can promote protein folding to alleviate ER stress. The bile acid tauroursodeoxycholic acid (TUDCA), has been described as a chemical chaperone. While promising in models of protein folding diseases, TUDCA's mechanism of action remains unclear. Here, we found TUDCA can rescue growth of yeast treated with the ER stressor tunicamycin (Tm), even in the absence of a functional UPR. In contrast, TUDCA failed to rescue growth on other ER stressors. Nor could TUDCA attenuate chronic UPR associated with specific gene deletions or over-expression of a misfolded mutant secretory protein. Neither pretreatment with or delayed addition of TUDCA conferred protection against Tm. Importantly, attenuation of Tm-induced toxicity required TUDCA's critical micelle forming concentration, suggesting a mechanism where TUDCA directly sequesters drugs. Indeed, in several assays, TUDCA treated cells closely resembled cells treated with lower doses of Tm. In addition, we found TUDCA can inhibit dyes from labeling intracellular compartments. Thus, our study challenges the model of TUDCA as a chemical chaperone and suggests that TUDCA decreases drug bioavailability, allowing cells to adapt to ER stress.

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