Biology subjects

Ellisman, M.

Publications and source records attributed to Ellisman, M..

At least 19 recordsLinked to original sources

bioRxiv2026

The circadian system is affected by Alzheimers disease independently from amyloid beta deposits

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Astrocyte-driven small vessel disease is an early, amyloid-independent feature of PSEN1 E280A familial Alzheimer's disease

Cerebral Small vessel disease (cSVD) is a prevalent feature of Alzheimers disease (AD) pathology. Whether this pathology is a late consequence of amyloid and tau accumulation or an early, direct effect of PSEN1 dysfunction has remained unresolved. We found that it is more severe in familial AD (FAD) caused by E280A mutation in presenilin 1 (PSEN1). These cases present with a distinctive proteomic signature, associated with pathological features, more dysregulated in the occipital cortex (OC) compared to the frontal cortex (FC), and characterized by multiple dysregulated proteins involved in extracellular matrix (ECM) and RNA-associated processes. This proteomic fingerprint was associated with abnormal collagen build up, ECM disorganization, and signatures of aberrant angiogenesis. Six months old transgenic knock-in mice homozygous for Psen1 E280A mutation (PSEN1Ki) also showed a similar phenotype with microvascular tortuosity and proteomic changes. Critically, these mice develop neither A{beta} plaques nor tau tangles, indicating that the shared microvascular and RNA-associated changes are direct consequences of PSEN1 dysfunction rather than downstream effects of amyloid pathology. Remarkably, dysregulated RNA-associated protein networks overlapped between FAD and PSEN1Ki mice. Cerebral microvessels microstructure in PSEN1Ki mice at two months and six months showed abnormal astrocytic end-feet with lamellar deposits implicating blood-brain barrier damage. Finally, single nuclei transcriptomic analysis of AD patients and controls showed similar abnormal astrocytes in both sporadic and familial variants, but FAD astrocytes expressed dysregulated genes identified in the proteomic analyses, such as GLUL, APOE, and CLU. Our findings suggest that cSVD is an early pathological event in PSEN1 FAD and that is driven by abnormal RNA-associated processes and astrocytic dysfunction.

neuroscience↗

bioRxiv2025

RPE pseudopods maintain photoreceptor outer segment renewal despite subretinal space expansion in Adam9 knockout mice

Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9 knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light-responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE "pseudopods" extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

cell biology↗

bioRxiv2025

Calorie Restriction modulates beta cell IP3R activity to regulate Ca2+ homeostasis and cell network connectivity

Calorie restriction (CR) promotes beta cell longevity by regulating cell identity, organelle and protein homeostasis, and metabolism pathways. CR beta cells have higher cAMP levels and mitochondria with an elevated potential to generate ATP. However, CR beta cells have reduced insulin secretion due to increased peripheral insulin sensitivity. How CR impacts beta cell Ca2+ homeostasis to regulate beta cell insulin release remains unknown. We investigated this question using acute pancreatic tissue slices prepared from ad-libitum (AL) or CR mice loaded with a low affinity Ca2+ indicator and recorded cytosolic Ca2+ gradients with fast confocal imaging. We exposed these slices to increasing glucose concentrations and applied our semi-automatic analysis pipeline to detect thousands of individual beta cells followed by identification of individual Ca2+ spiking events. We observed that CR beta cells have fast short-amplitude Ca2+ oscillations that correlate with largely disconnected beta cell networks across the islet. Using acetylcholine stimulation, we found that faster IP3R-driven Ca2+ oscillations linked to higher cytosolic cAMP levels protect beta cells against acute depletion of ER Ca2+ stress. Therefore, this study provides new mechanistic insight into adaptation of beta cell and of beta cell networks to CR interventions. Article highlightsO_LIBeta cells from calorie restricted (CR) mice have decreased insulin release, however the mechanisms underlying this adaptive response remain unknown. C_LIO_LICR beta cells have elevated basal cytosolic cAMP ([cAMP]cyt) compared to beta cells in control ad libitum fed (AL) mice, and they operate with faster and shorter cytosolic Ca2+ oscillations. C_LIO_LIWhile AL beta cells form interconnected activity networks, CR beta cells are largely disconnected and fire more independently of each other. C_LIO_LIIslets of CR mice can sustain prolonged activity during ER stressing conditions due to elevated IP3R activity and improved Ca2+ homeostasis. C_LI Why did we undertake this study?We have previously shown that calorie restriction (CR) promotes beta cell longevity by enhancing beta cell identity and organelle homeostasis mechanisms. This long-lived phenotype correlated with the onset of enhanced peripheral insulin sensitivity and reduced beta cell insulin release in vivo despite higher cAMP levels and increased potential for mitochondrial ATP generation. However, the mechanisms underlying the reduced cell insulin release phenotype of CR beta cells remains unknown. Therefore, we investigated the underlying Ca2+ homeostasis mechanisms regulating insulin release in AL and CR beta cells. What is the specific question(s) we wanted to answer?We were interested in determining what are the cell Ca2+ activity patterns during basal and glucose-stimulated conditions in AL and CR beta cells. In addition, we also investigated how CR beta cells respond to epinephrine inhibition and supra-stimulatory concentrations of acetylcholine (ACh), which drive acute beta cell stress by disrupting normal cAMP and ER Ca2+ signaling, respectively. Finally, we investigate whether CR beta cells formed more interconnected beta cell networks driven by changes in Ca2+ activity patterns. What did we find?We found that CR beta cells are more active with significantly higher rates of Ca2+ oscillation at basal and high glucose concentrations. In fact, CR beta cells have shorter inter-Ca2+ event intervals that are more resistant to depletion of cAMP by epinephrine application. In contrast, stimulation of IP3R activity (to force depletion of ER Ca2+ stores) by supraphysiological ACh concentrations revealed that CR beta cells were able to sustain a prolonged Ca2+ activity versus AL beta cells. Surprisingly, this enhanced beta cell activity profile reduced beta cell activity network connectivity. What are the implications of our findings?Our work demonstrates that CR beta cells have higher baseline and glucose-stimulated Ca2+ activity due to higher cAMP levels. These cells also have dominant IP3R activity that grants improved ER Ca2+ homeostasis and significantly reduces beta cell network connectivity to tone down insulin secretion. These studies provide a mechanistic understanding of how beta cells adapt to CR and to CR-associated enhanced insulin sensitivity.

cell biology↗

bioRxiv2025

Development of a Charge-Resistant Embedding Media for High-Performance Serial Block Face Imaging of Cells and Tissues

Serial block-face scanning electron microscopy (SBEM) enables high-resolution 3D imaging of biological specimens but is often limited by specimen charging due to the use of non-conductive epoxy resins. While heavy metal staining and variable-pressure SEM can reduce charging, these methods compromise resolution or are only partially effective. We report a novel approach using polyethylene glycol (PEG 3350), a water-soluble, non-conductive polymer, as a doping agent to reduce specimen charging without sacrificing imaging quality. Although PEG 3350 alone lacks the mechanical and sectioning properties required for SBEM, we found it can be dissolved in standard Durcupan epoxy resin to improve charge resistance while maintaining physical integrity. Resins doped with 10% PEG 3350 demonstrated a substantial reduction in charging at 1.4-1.8 keV in charge-prone samples such as cultured HeLa cells, lung, and brain tissues, while retaining transparency and sectionability. Unlike other methods, the doped resin performs in high vacuum without compromising signal-to-noise ratio or spatial resolution. Notably, the PEG-doped resin reduced charging at voltages above the typical threshold for undoped resins, which show artifacts above 1.0 keV. Though some charging persisted in lung tissue at 2.0 keV, near-complete mitigation was achieved at slightly lower voltages. Our findings suggest that PEG-doped resins provide a promising route to minimize specimen charging in SBEM, and future studies optimizing PEG molecular weight and concentration could yield a universally charge-resistant embedding medium compatible with high-resolution imaging across diverse sample types.

cell biology↗

bioRxiv2025

Population-level morphological analysis of paired CO2- and odor-sensing olfactory neurons in D. melanogaster via volume electron microscopy

Dendritic morphology is a defining characteristic of neuronal subtypes. In Drosophila, heterotypic olfactory receptor neurons (ORNs) expressing different receptors display diverse dendritic morphologies, but whether such diversity exists among homotypic ORNs remains unclear. Using serial block-face scanning electron microscopy on cryofixed tissues, we analyzed the majority of CO2-sensing neurons (ab1C) and their odor-sensing neighbors (ab1D) in the D. melanogaster antenna. Surprisingly, ab1C neurons featured flattened, sheet-like dendrites--distinct from the cylindrical branches typical of odor-sensing neurons--and displayed remarkable diversity, ranging from plain sheets to tube-like structures that enclose several neighboring dendrites, forming "dendrite-within-dendrite" structures. Similarly, ab1D dendrites varied from simple, unbranched forms to numerously branched morphologies. These findings suggest that morphological heterogeneity is common even among homotypic ORNs, potentially expanding their functional adaptability and ranges of sensory physiological properties.

neuroscience↗

bioRxiv2024

Ultrastructural correlates of circadian structural plasticity

In Drosophila, about 250 clock neurons in the brain form a network that orchestrates circadian rhythmicity. Among them, eight small Lateral ventral Neurons (s-LNvs) play a critical role, synchronizing the circadian ensemble via the neuropeptide Pigment-Dispersing Factor (PDF). Moreover, their neurites show daily variations in morphology, PDF levels, synaptic markers and connectivity. This process, called circadian structural plasticity, is ill-defined at the subcellular level. Here, we present 3D volumes of the s-LNv terminals generated by Serial Block-face Scanning Electron Microscopy (SBEM) at three key time points, two hours before lights-ON, two hours after lights-ON, and two hours after lights-OFF. We report a reduction in the number of neuronal varicosities at night, which reflects (and probably regulates) the cycling of the components we found therein. Indeed, in the morning we observed more presynaptic sites and increased accumulation and release of dense core vesicles. These rhythms were paralleled by periodic changes in mitochondrial structure that suggest daily modulation of their activity. We propose that circadian plasticity of the functionally relevant structures within presynaptic varicosities cyclically modulates the influence of the s-LNvs on the clock network.

neuroscience↗

bioRxiv2024

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

biochemistry↗

bioRxiv2024

Human rod photoreceptor outer segments are supported by accessory inner segment structures

The first steps in vision take place in photoreceptor cells, which are highly compartmentalized neurons exhibiting significant structural variation across species. The light-sensitive ciliary compartment, called the outer segment, is located atop of the cell soma, called the inner segment. In this study, we present an ultrastructural analysis of human photoreceptors, which reveals that, in contrast to this classic arrangement, the inner segment of human rods extends alongside the outer segment to form a structure hereby termed the "accessory inner segment". While reminiscent of the actin-based microvilli known as "calyceal processes" observed in other species, the accessory inner segment is a unique structure: (1) it contains an extensive microtubule-based cytoskeleton, (2) it extends far alongside the outer segment, (3) its diameter is comparable to that of the outer segment, (4) it contains numerous mitochondria, and (5) it forms electron-dense structures that likely mediate adhesion to the outer segment. Given that the spacing of extrafoveal human photoreceptors is more sparse than in non-primate species, with vast amounts of interphotoreceptor matrix present between cells, the closely apposed accessory inner segment likely provides structural support to the outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.

cell biology↗

bioRxiv2024

ER-phagy drives age-onset remodeling of endoplasmic reticulum structure-function and lifespan

The endoplasmic reticulum (ER) comprises an array of structurally distinct subdomains, each with characteristic functions. While altered ER-associated processes are linked to age-onset pathogenesis, whether shifts in ER morphology underlie these functional changes is unclear. We report that ER remodeling is a conserved feature of the aging process in models ranging from yeast to C. elegans and mammals. Focusing on C. elegans as an exemplar of metazoan aging, we find that as animals age, ER mass declines in virtually all tissues and ER morphology shifts from rough sheets to tubular ER. The accompanying large-scale shifts in proteomic composition correspond to the ER turning from protein synthesis to lipid metabolism. To drive this substantial remodeling, ER-phagy is activated early in adulthood, promoting turnover of rough ER in response to rises in luminal protein-folding burden and reduced global protein synthesis. Surprisingly, ER remodeling is a pro-active and protective response during aging, as ER-phagy impairment limits lifespan in yeast and diverse lifespan-extending paradigms promote profound remodeling of ER morphology even in young animals. Altogether our results reveal ER-phagy and ER morphological dynamics as pronounced, underappreciated mechanisms of both normal aging and enhanced longevity.

cell biology↗

bioRxiv2024

Scalable electron tomography for connectomics

We demonstrate limited-tilt, serial section electron tomography (ET), which can non-destructively map brain circuits over large 3D volumes and reveal high-resolution, supramolecular details within subvolumes of interest. We show accelerated ET imaging of thick sections (>500 nm) with the capacity to resolve key features of neuronal circuits including chemical synapses, endocytic structures, and gap junctions. Furthermore, we systematically assessed how imaging parameters affect image quality and speed to enable connectomic-scale projects.

neuroscience↗

bioRxiv2024

Synaptic architecture of a memory engram in the mouse hippocampus

Memory engrams are formed through experience-dependent remodeling of neural circuits, but their detailed architectures have remained unresolved. Using 3D electron microscopy, we performed nanoscale reconstructions of the hippocampal CA3-CA1 pathway following chemogenetic labeling of cellular ensembles with a remote history of correlated excitation during associative learning. Projection neurons involved in memory acquisition expanded their connectomes via multi-synaptic boutons without altering the numbers and spatial arrangements of individual axonal terminals and dendritic spines. This expansion was driven by presynaptic activity elicited by specific negative valence stimuli, regardless of the co-activation state of postsynaptic partners. The rewiring of initial ensembles representing an engram coincided with local, input-specific changes in the shapes and organelle composition of glutamatergic synapses, reflecting their weights and potential for further modifications. Our findings challenge the view that the connectivity among neuronal substrates of memory traces is governed by Hebbian mechanisms, and offer a structural basis for representational drifts.

neuroscience↗

bioRxiv2023

Glaucoma-associated Optineurin mutations increase transmitophagy in a vertebrate optic nerve

We previously described a process referred to as transmitophagy where mitochondria shed by retinal ganglion cell (RGC) axons are transferred to and degraded by surrounding astrocytes in the optic nerve head of mice. Since the mitophagy receptor Optineurin (OPTN) is one of few large- effect glaucoma genes and axonal damage occurs at the optic nerve head in glaucoma, here we explored whether OPTN mutations perturb transmitophagy. Live-imaging of Xenopus laevis optic nerves revealed that diverse human mutant but not wildtype OPTN increase stationary mitochondria and mitophagy machinery and their colocalization within, and in the case of the glaucoma-associated OPTN mutations also outside of, RGC axons. These extra-axonal mitochondria are degraded by astrocytes. Our studies support the view that in RGC axons under baseline conditions there are low levels of mitophagy, but that glaucoma-associated perturbations in OPTN result in increased axonal mitophagy involving the shedding and astrocytic degradation of the mitochondria. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/542507v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@c1d3d4org.highwire.dtl.DTLVardef@13a33e6org.highwire.dtl.DTLVardef@2544beorg.highwire.dtl.DTLVardef@c456a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗

bioRxiv2021

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpus itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 required PTPN23 but not the retromer subunits. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23. Author SummaryVpu triggers the degradation or mis-localization of proteins important to the hosts immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the proteins location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpus presence at the limiting membranes of multi-vesicular bodies.

microbiology↗

bioRxiv2020

Spatially-resolved correlative microscopy and microbial identification reveals dynamic depth- and mineral-dependent anabolic activity in salt marsh sediment

Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural-functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7-day in situ incubations with a non-canonical amino acid that was incorporated into new biomass, samples were embedded and analyzed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence-activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from [~]60% in the top cm to 10-25% between 2-7 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to sulfate-reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non-quartz minerals including rutile, orthoclase, and plagioclase. Microbe-mineral relationships appear to be dynamic and context-dependent arbiters of biogeochemical cycling. Statement of SignificanceMicroscale spatial relationships dictate critical aspects of a microbiomes inner workings and emergent properties, such as evolutionary pathways, niche development, and community structure and function. However, many commonly used methods in microbial ecology neglect this parameter - obscuring important microbe-microbe and microbe-mineral interactions - and instead employ bulk-scale methodologies that are incapable of resolving these intricate relationships. This benchmark study presents a compelling new approach for exploring the anabolic activity of a complex microbial community by mapping the precise spatial configuration of anabolically active organisms within mineralogically heterogeneous sediment through in situ incubation, resin embedding, and correlative fluorescence and electron microscopy. In parallel, active organisms were identified through fluorescence-activated cell sorting and 16S rRNA gene sequencing, enabling a powerful interpretive framework connecting location, identity, activity, and putative biogeochemical roles of microbial community members. We deploy this novel approach in salt marsh sediment, revealing quantitative insights into the fundamental principles that govern the structure and function of sediment-hosted microbial communities. In particular, at different sediment horizons, we observed striking changes in the proportion of anabolically active cells, the identities of the most prominent active community members, and the nature of microbe-mineral affiliations. Improved approaches for understanding microscale ecosystems in a new light, such as those presented here, reveal environmental parameters that promote or constrain metabolic activity and clarify the impact that microbial communities have on our world.

microbiology↗

bioRxiv2020

Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets

Clathrin has established roles in endocytosis, with clathrin-cages enclosing membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin synthesized in cell-bodies is conveyed into axons and synapses via slow axonal transport; as shown by classic pulse-chase radiolabeling. What is the cargo-structure, and mechanisms underlying transport and presynaptic-targeting of clathrin? What is the precise organization at synapses? Combining live-imaging, mass-spectrometry (MS), Apex-labeled EM-tomography and super-resolution, we found that unlike dendrites where clathrin transiently assembles/disassembles as expected, axons contain stable transport-packets that move intermittently with an anterograde bias; with actin/myosin-VI as putative tethers. Transport-packets are unrelated to endocytosis, and the overall kinetics generate a slow biased flow of axonal clathrin. Synapses have integer-numbers of clathrin-packets circumferentially abutting the synaptic-vesicle cluster, advocating a model where delivery of clathrin-packets by slow axonal transport generates a radial organization of clathrin at synapses. Our experiments reveal novel trafficking mechanisms, and an unexpected nanoscale organization of synaptic clathrin.

neuroscience↗

bioRxiv2019

Class IIa HDACs regulate learning and memory through dynamic experience-dependent repression of transcription.

The formation of new memories requires transcription. However, the mechanisms that limit signaling of relevant gene programs in space and time for precision of coding and the roles of these programs in specific aspects of information processing remain poorly understood. We found that, during learning, the cellular patterns of expression of early response genes (ERGs) are regulated by class IIa HDACs 4 and 5, transcriptional repressors that transiently enter neuronal nuclei from cytoplasm after sensory input. Mice lacking these repressors in the forebrain have abnormally broad experience-dependent expression of ERGs, altered synaptic architecture and function, elevated anxiety, and severely impaired memory. By acutely manipulating the nuclear activity of class IIa HDACs in behaving animals using a chemical-genetic technique, we further demonstrate that rapid induction of ERG programs is critical for memory acquisition but these programs may become dispensable when a stable memory is formed. These results provide new insights into the molecular basis of memory storage.

neuroscience↗