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3836 Publications

Showing 11-20 of 3836 results
02/21/24 | RAL-1 signaling regulates lipid composition in .
Wu Y, Lee M, Mutlu AS, Wang M, Reiner DJ
MicroPubl Biol. 2024-02-21;2024:. doi: 10.17912/micropub.biology.001054

Signaling by the Ral small GTPase is poorly understood . animals with constitutively activated RAL-1 or deficient for the inhibitory RalGAP, HGAP-1 /2, display pale intestines. Staining with Oil Red O detected decreased intestinal lipids in the deletion mutant relative to the wild type. Constitutively activated RAL-1 decreased lipid detected by stimulated Raman scattering (SRS) microscopy, a label-free method of detecting lipid by laser excitation and detection. A signaling-deficient missense mutant for RAL-1 also displayed reduced lipid staining via SRS. We conclude that RAL-1 signaling regulates lipid homeostasis, biosynthesis or storage in live animals.

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03/11/24 | Spot Spine, a freely available ImageJ plugin for 3D detection and morphological analysis of dendritic spines
Gilles J, Mailly P, Ferreira T, Boudier T, Heck N
F1000Research. 2024-03-11;13:. doi: 10.12688/f1000research.146327.1

Background

Dendritic spines are tiny protrusions found along the dendrites of neurons, and their number is a measure of the density of synaptic connections. Altered density and morphology is observed in several pathologies, and spine formation as well as morphological changes correlate with learning and memory. The detection of spines in microscopy images and the analysis of their morphology is therefore a prerequisite for many studies. We have developed a new open-source, freely available, plugin for ImageJ/FIJI, called Spot Spine, that allows detection and morphological measurements of spines in three dimensional images.

Method

Local maxima are detected in spine heads, and the intensity distribution around the local maximum is computed to perform the segmentation of each spine head. Spine necks are then traced from the spine head to the dendrite. Several parameters can be set to optimize detection and segmentation, and manual correction gives further control over the result of the process.

Results

The plugin allows the analysis of images of dendrites obtained with various labeling and imaging methods. Quantitative measurements are retrieved including spine head volume and surface, and neck length.

Conclusion

The plugin and instructions for use are available at https://imagej.net/plugins/spot-spine.

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03/12/24 | Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)
Talon Chandler , Min Guo , Yijun Su , Jiji Chen , Yicong Wu , Junyu Liu , Atharva Agashe , Robert S. Fischer , Shalin B. Mehta , Abhishek Kumar , Tobias I. Baskin , Valentin Jamouille , Huafeng Liu , Vinay Swaminathan , Amrinder Nain , Rudolf Oldenbourg , Patrick La Riviere , Hari Shroff
bioRxiv. 2024-03-12:. doi: 10.1101/2024.03.09.584243

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.Competing Interest StatementH.S., A.K., S.M., P.L.R., R.O., Y.W., and T.C. hold US Patent #11428632.

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03/12/24 | Three-dimensional spatio-angular fluorescence microscopy with a polarized dual-view inverted selective-plane illumination microscope (pol-diSPIM)
Talon Chandler , Min Guo , Yijun Su , Jiji Chen , Yicong Wu , Junyu Liu , Atharva Agashe , Robert S. Fischer , Shalin B. Mehta , Abhishek Kumar , Tobias I. Baskin , Valentin Jamouille , Huafeng Liu , Vinay Swaminathan , Amrinder Nain , Rudolf Oldenbourg , Patrick La Riviere , Hari Shroff
bioRxiv. 2024-03-12:. doi: 10.1101/2024.03.09.584243

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the three-dimensional orientations and diffraction-limited positions of ensembles of fluorescent dipoles that label biological structures, and we share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model our samples, their excitation, and their detection using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labelled giant unilamellar vesicles, fast-scarlet-labelled cellulose in xylem cells, and phalloidin-labelled actin in U2OS cells. Additionally, we observe phalloidin-labelled actin in mouse fibroblasts grown on grids of labelled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.Competing Interest StatementH.S., A.K., S.M., P.L.R., R.O., Y.W., and T.C. hold US Patent #11428632.

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03/07/24 | Dendritic voltage imaging maps the biophysical basis of plateau potentials in the hippocampus
Pojeong Park , J. David Wong-Campos , Daniel Itkis , Byung Hun Lee , Yitong Qi , Hunter C. Davis , Jonathan B. Grimm , Sarah E. Plutkis , Luke Lavis , Adam Ezra Cohen
bioRxiv. 2024-03-07:. doi: 10.1101/2023.06.02.543490

Dendrites on neurons integrate synaptic inputs to determine spike timing. Dendrites also convey back-propagating action potentials (bAPs) which interact with synaptic inputs to produce plateau potentials and to mediate synaptic plasticity. The biophysical rules which govern the timing, spatial structures, and ionic character of dendritic excitations are not well understood. We developed molecular, optical, and computational tools to map sub-millisecond voltage dynamics throughout the dendritic trees of CA1 pyramidal neurons under diverse optogenetic and synaptic stimulus patterns, in acute brain slices. We observed history-dependent bAP propagation in distal dendrites, driven by locally generated Na+ spikes (dSpikes). Dendritic depolarization creates a transient window for dSpike propagation, opened by A-type KV channel inactivation, and closed by slow NaV inactivation. Collisions of dSpikes with synaptic inputs triggered calcium channel and N-methyl-D-aspartate receptor (NMDAR)-dependent plateau potentials, with accompanying complex spikes at the soma. This hierarchical ion channel network acts as a spike-rate accelerometer, providing an intuitive picture of how dendritic excitations shape associative plasticity rules.Competing Interest StatementThe authors have declared no competing interest.

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03/01/24 | Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast
Daniel J. Barrero , Sithara S. Wijeratne , Xiaowei Zhao , Grace F. Cunningham , Rui Yan , Christian R. Nelson , Yasuhiro Arimura , Hironori Funabiki , Charles L. Asbury , Zhiheng Yu , Radhika Subramanian , Sue Biggins
bioRxiv. 2024-03-01:. doi: 10.1101/2024.02.28.582571

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

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03/06/24 | Assessing the impact of Brd2 depletion on chromatin compartmentalization
Advait Athreya , Liangqi Xie , Robert Tjian , Bin Zhang , Zhe J. Liu
bioRxiv. 2024-03-06:. doi: 10.1101/2024.03.02.583085

Recent insights into genome organization have emphasized the importance of A/B chromatin compartments. While our previous research showed that Brd2 depletion weakens compartment boundaries and promotes A/B mixing 1, Hinojosa-Gonzalez et al.2 were unable to replicate the findings. In response, we revisited our Micro-C data and successfully replicated the original results using the default parameters in the cooltools software package. We show that, after correcting inconsistencies with the selection and phasing of the compartment profiles, the decrease in B compartment strength persists but the change in compartment identity is to a much lesser extent than originally reported. To further assess the regulatory role of Brd2, we used saddle plots to determine the strength of compartmentalization and observed a consistent decrease of compartment strength especially at B compartments upon Brd2 depletion. This study highlights the importance of selecting appropriate parameters and analytical tools for compartment analysis and carefully interpreting the results.

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Integrative Imaging
03/04/24 | Four-dimensional quantitative analysis of cell plate development using lattice light sheet microscopy identifies robust transition points between growth phases.
Sinclair R, Wang M, Jawaid MZ, Longkumer T, Aaron J, Rossetti B, Wait E, McDonald K, Cox D, Heddleston J, Wilkop T, Drakakaki G
Journal of Experimental Botany. 2024-03-04:. doi: 10.1093/jxb/erae091

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodeling, and timely polysaccharide deposition, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM) we studied cell plate development in four dimensions, through the behavior of the cytokinesis specific GTPase YFP-RABA2a vesicles. We monitored the entire length of cell plate development, from its first emergence, with the aid of YFP-RABA2a, both in the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable, cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

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02/27/24 | Haploidy-linked cell proliferation defects limit larval growth in Zebrafish
Kan Yaguchi , Daiki Saito , Triveni Menon , Akira Matsura , Takeomi Mizutani , Tomoya Kotani , Sreelaja Nair , Ryota Uehara
bioRxiv. 2024-02-27:. doi: 10.1101/2022.05.12.491746

Haploid larvae in non-mammalian vertebrates are lethal with characteristic organ growth retardation collectively called “haploid syndrome.” In contrast to mammals whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 significantly improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are critical cellular causes that limit the larval growth in the haploid state, potentially placing an evolutionary constraint on allowable ploidy status in the non-mammalian vertebrate life cycle.

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02/28/24 | High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.
Hario S, Le GN, Sugimoto H, Takahashi-Yamashiro K, Nishinami S, Toda H, Li S, Marvin JS, Kuroda S, Drobizhev M, Terai T, Nasu Y, Campbell RE
ACS Central Science. 2024-02-28;10(2):402-416. doi: 10.1021/acscentsci.3c01250

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (Δ/ = 15 to 30 ), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an preparation of brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

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