Biology subjects

Guzman, E.

Publications and source records attributed to Guzman, E..

8 recordsLinked to original sources

bioRxiv2024

Untangling structural molecular details of the endocytic adaptor protein CALM upon binding with phosphatidylinositol 4,5-bisphosphate-containing model membranes

Clathrin assembly lymphoid myeloid leukemia protein (CALM) is involved in the formation of clathrin-mediated endocytic coats by virtue of binding many proteins involved in the process, including clathrin itself and AP2 cargo adaptor complex. CALM is able to specifically recognize the inner leaflet of the plasma membrane by binding the membranes phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Here, a biophysical approach, primarily using neutron and X-ray scattering and solid-state NMR experiments, was exploited to investigate CALM interaction with PtdIns(4,5)P2-presenting model membranes. The presented experimental data reveal how the CALM folded domain is partly accommodated within the lipid membrane, directly interacting with PtdIns(4,5)P2 phosphates. Moreover, these data suggest that CALMs amphiphilic N-terminal helix buries into the membrane, not only stabilising the protein docking to the membrane but also providing a mechanism to induce membrane curvature.

biophysics↗

bioRxiv2022

Automated detection of extracellular action potentials from single neurons

Multi-electrode arrays (MEAs) non-invasively record extracellular action potentials (eAPs, also known as spikes) from hundreds of neurons simultaneously. However, because extracellular electrodes sample from the local electrical field, each electrode can detect eAPs from multiple nearby neurons. Interpreting spike trains at individual electrodes of high-density arrays requires spike sorting, a computational process which groups eAPs from single units based on assumptions of how spike waveforms correlate with different neuronal sources. Additionally, when experimental conditions result in changes to eAP waveforms, spike sorting routines may have difficulty correlating eAPs from multiple neurons at single electrodes before and after such waveform changes. We present here a novel, empirical method for unambiguously isolating eAPs from individual, uniquely identifiable neurons, based on automated multi- point detection of action potential propagation. This method is insensitive to changes in eAP waveform morphology because it makes no assumptions about the relationship between spike waveform and neuronal source. Our algorithm for automated detection of action potential propagation produces a fingerprint that uniquely identifies those spikes from each neuron. By unambiguously isolating eAPs from multiple neurons in each recording, on a range of platforms and experimental preparations, our method now enables high-content screening with contemporary MEAs. We outline the limitations and strengths of propagation-based isolation of eAPs from single neurons and propose how our automated method complements spike sorting and could be adapted to in vivo use.

neuroscience↗

bioRxiv2021

Intrinsic network activity in human brain organoids

Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiological behavior of neuronal circuits within organoids remains relatively under-explored. With high-density CMOS microelectrode arrays (26,400 electrodes) and shank electrodes (960 electrodes), we probed broadband and three-dimensional extracellular field recordings generated by spontaneous activity of human brain organoids. These recordings simultaneously captured local field potentials (LFPs) and single-unit activity extracted through spike sorting. From spiking activity, we estimated a directed functional connectivity graph of synchronous neural network activity, which showed a large number of weak functional connections enmeshed within a network skeleton of significantly fewer strong connections. Treatment of the organoid with a benzodiazepine induced a reproducible signature response that shortened the inter-burst intervals, increased the uniformity of the firing pattern within each burst and decreased the population of weakly connected edges. Simultaneously examining the spontaneous LFPs and their phase alignment to spiking showed that spike bursts were coherent with theta oscillations in the LFPs. Our results demonstrate that human brain organoids have self-organized neuronal assemblies of sufficient size, cellular orientation, and functional connectivity to co-activate and generate field potentials from their collective transmembrane currents that phase-lock to spiking activity. These results point to the potential of brain organoids for the study of neuropsychiatric diseases, drug mechanisms, and the effects of external stimuli upon neuronal networks.

neuroscience↗

bioRxiv2020

Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signalling

Viruses routinely employ strategies to prevent the activation of innate immune signalling in infected cells. RSV is no exception, encoding two accessory proteins (NS1 and NS2) which are well established to block Interferon signalling. However, RSV-encoded mechanisms for inhibiting NF-{kappa}B signalling are less well characterised. In this study we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins, and indeed distinct from other known viral mechanisms of NF-{kappa}B inhibition. In both human and bovine RSV infected cells we demonstrated that the P65 subunit of NF-{kappa}B is rerouted to perinuclear puncta in the cytoplasm, puncta which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured P65 was unable to translocate to the nucleus or transactivate a NF-{kappa}B reporter following TNF- stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalise RSV N protein and P65 within bRSV IBs; granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterisation of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signalling which relies on sequestration of the NF-{kappa}B subunit p65 to a biomolecular condensate - a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells. Impact summaryMany viruses replicate almost entirely in the cytoplasm of infected cells, without too many direct interactions with the nucleus. Examples include respiratory syncytial virus (RSV), measles, Ebola and Nipah; however, how these pathogens are able to compartmentalise their life cycle to provide favourable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterised. In this paper we show that bovine RSV (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. These organelles are unusually membrane-less; likely forming by a process called liquid-liquid phase separation which involves macro-molecular interactions between the viral proteins N and P. We also showed that these organelles, otherwise known as inclusion bodies (IBs), are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signalling processes that tell the nucleus the cell is infected. Using fluorescent bioimaging and a combination of confocal and electron microscopy we then characterised this interaction in detail, also confirming that human RSV (hRSV) employs the same mechanism. Like hRSV, bRSV viral RNA replication also takes place in the IB, likely meaning these organelles are a functionally conserved feature of orthopneumoviruses.

microbiology↗

bioRxiv2018

Farnesyl Transferase Inhibition for the Treatment of Tauopathies

Tau inclusions are a shared feature of many neurodegenerative conditions and tau mutations lead to frontotemporal dementia. Approaches to treatment of these conditions have focused directly on the tau protein by targeting its post-translational modifications, its levels and its tendency to aggregate. We discovered a novel regulatory pathway for tau degradation that operates through the Rhes protein, a GTPase. Rhes is farnesylated and treatment with the farnesyl transferase inhibitor, lonafarnib, reduced Rhes, attenuated behavioral abnormalities, significantly reduced atrophy, tau inclusions, sumoylation and ubiquitination, as well as microgliosis in the rTg4510 tauopathy mouse. Direct reduction of Rhes levels reproduced the results observed with lonafarnib. The mechanism of lonafarnib action, as mediated by Rhes to reduce tau pathology, operates through the lysosome without involvement of the proteasome. Finally we show that the developmental increase in Rhes levels can be homeostatically regulated in the presence of tau mutations as a protective mechanism through which cells sense abnormal tau before any pathology is present. The extensive human trials of lonafarnib for other conditions, makes this drug ideal for repurposing to treat tauopathies.\n\nOne-sentence summaryVia a mechanism that involves targeting Rhes, lonafarnib can induce lysosomal-mediated tau degradation and prevent pathology in a tau mouse model

neuroscience↗

bioRxiv2017

Enhanced neuronal regeneration in the CAST/Ei mouse strain is linked to expression of differentiation markers after injury

Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei specific upregulation of Ascl1 after injury. Ascl1 overexpression in C57BL/6 mice DRG neurons enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is down-regulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single cell RNAseq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury.

molecular biology↗

bioRxiv2017

RNA Stores Tau Reversibly in Complex Coacervates

Non-membrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable {beta}-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and pre-dispose the protein toward the formation of insoluble fibrils.

biophysics↗

bioRxiv2017

A Molecular Signature for Anastasis, Recovery from the Brink of Apoptotic Cell Death

During apoptosis, executioner caspase activity has been considered a point of no return. However, recent studies show that cells can survive caspase activation following transient apoptotic stimuli, a process named anastasis. To identify a molecular signature, we performed whole transcriptome RNA sequencing of untreated, apoptotic, and recovering HeLa cells. We found that anastasis is an active, two-stage program. During the early stage, cells transition from growth-arrested to growing. In the late stage, cells change from proliferating to migratory. Strikingly, some early recovery mRNAs were elevated first during apoptosis, implying that dying cells poise to recover, even while still under apoptotic stress. Furthermore, TGF{beta}-induced Snail expression is required for anastasis, and recovering cells exhibit prolonged elevation of pro-angiogenic factors. This study demonstrates similarities in the anastasis genes, pathways, and cell behaviors to those activated in wound healing. This study identifies a repertoire of potential targets for therapeutic manipulation of this process.

cell biology↗