Biology subjects

D'Este, E.

Publications and source records attributed to D'Este, E..

15 recordsLinked to original sources

bioRxiv2026

Paralemmin-2 is a membrane-anchored cytoskeletal constituent of Axon Initial Segments and nodes of Ranvier

The axon initial segment (AIS) and nodes of Ranvier (NoR) are essential for action potential initiation and propagation. They share many features of their molecular architecture, and their assembly mechanisms converge on the membrane-associated periodic skeleton (MPS). Here, we identify Paralemmin-2 (Palm2) as a component of both the AIS and NoR. Palm2 depletion shortens the AIS and reduces neuronal excitability. Endogenous Palm2 in the AIS is non-periodic, but upon overexpression it associates with the MPS, localizing to actin rings and reducing {beta}IV-spectrin abundance and periodicity. In NoR of the central and peripheral nervous system, Palm2 localizes to different subdomains - nodal or paranodal, respectively. Palm2, and its homolog Palm1, bind the deubiquitinase USP7, implicating paralemmins in proteostasis at the MPS. The complementary localizations of Palm2 and Palm1 at the AIS/NoR or axon shafts, respectively, parallel the distributions of {beta}-spectrin and ankyrin isoforms between these axonal compartments. We propose that Palm2 modulates the submembrane cytoskeleton and its membrane attachment, and thus contributes to the assembly, functioning and remodeling of the AIS and NoR.

cell biology↗

bioRxiv2025

Controlling the Formation of Multiple Condensates in the Synapse

The postsynaptic density (PSD) is a molecule rich structure that continuously adapts its organization controlling synaptic physiology and transmission. Experimental studies have shown that this organization is dominated by the formation of condensates or (nano)clusters and by the seamless transition in their numbers as response to synaptic plasticity. In this study, we utilize different computational modeling frameworks to show that variations in the level of local protein concentrations together with the modulation of protein binding strengths can be key molecular factors in controlling the formation and number of clusters in the synapse. Comparison to MINFLUX data of spatial localization of PSD95 in the postsynapse under different activity conditions allowed us to derive predictions about the correlation between these two factors across a population of synapses. Co-variations of the factors, to mimic a simple LTP protocol, shows that a PSD containing a single cluster can reorganize to form multiple clusters that persists for long periods of time, providing a potential explanation of recent experimental data of PSD reorganization.

molecular biology↗

bioRxiv2024

Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton

The Membrane-associated Periodic Skeleton (MPS) is a specialized submembrane cytoskeleton of neuronal cells, characterized by a highly ordered 190 nm periodic lattice, with emerging functions in mechanical resilience, inter- and intracellular signaling, and action potential transmission. Here, we identify Paralemmin-1 (Palm1) as a new component and regulator of the MPS. Palm1 binds to the N-terminal region of {beta}II-spectrin, a core MPS component, and is periodically organized along the axon in hippocampal neurons. Applying the 3D imaging power of MINFLUX, we locate Palm1 in close proximity (<20 nm) to the actin-capping protein and MPS component adducin. Functionally, Palm1 overexpression enhances the degree of periodicity of several MPS proteins ({beta}II-spectrin, adducin, and ankyrinB) without altering their local concentrations, while the knock-out severely compromises the MPS structure and modifies electrophysiological properties of neurons. Both the MPS-binding and remodelling activities of Palm1 are abolished by mutating a single amino acid (W54A) in the conserved Paralemmin sequence motif. Our findings identify Palm1 as the first protein specifically dedicated to organizing the MPS, and will advance the understanding of the regulation of MPS assembly and remodelling, as well as of the Paralemmin protein family.

cell biology↗

bioRxiv2024

MINFLUX Reveals Dynein Stepping in Live Neurons

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism whereby dynein consumes one adenosine 5-triphosphate (ATP) per step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

biophysics↗

bioRxiv2023

Neuronal activity modulates the incorporation of newly translated PSD-95 into a robust structure as revealed by STED and MINFLUX

The postsynaptic density component PSD-95 undergoes activity-dependent plasticity mechanisms that rely on protein synthesis and structural remodeling. How synaptic activity can influence these dynamics at the single synapse level remains unclear. Here we combine genome-editing, pulse-chase experiments, STED and 3D MINFLUX nanoscopy on hippocampal neuronal cultures to study the integration of newly translated PSD-95 molecules at postsynaptic sites and their rearrangement within individual clusters at near-molecular resolution. We show that the amount of newly translated PSD-95 recruited to individual synapses scales with synaptic size, and modulates in a bidirectional manner, resulting in less new protein following excitatory and more new protein following inhibitory stimulation. Furthermore, we show that within synaptic clusters PSD-95 has a dispersed organization that is largely robust to long-lasting changes in activity. Altogether, this work sheds new light on the mechanisms underlying plasticity at the single synapse level, adding previously inaccessible information.

neuroscience↗

bioRxiv2023

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

neuroscience↗

bioRxiv2023

Intrinsic microtubule destabilization of multiciliated choroid plexus epithelial cells during postnatal lifetime

Choroid plexus (ChP) epithelium is composed of specialized multiciliated cells. By using multiple microscopic techniques, biochemical approaches in various mutant mice and longitudinal analysis from mouse embryogenesis to aging, we show that ChP cilia are built on a gradient of events which are spatio-temporally regulated. We uncover that ChP cilia develop prenatally since early tissue morphogenesis, and proceeds as a multi-step process characterized by basal body multiplication and axoneme formation directly at the apical cellular compartment. Our data also show that choroid plexus cilia contain both primary and motile features. Remarkably, we demonstrate that ChP cilia undergo axoneme resorption, starting from early youth, through a tubulin destabilization process, which is primarily controlled by polyglutamylation levels and could be mitigated by the removal of the microtubule-severing enzyme spastin. Notably, we demonstrate that this phenotype is preserved in human samples.

developmental biology↗

bioRxiv2022

Synaptic status differentially regulates neurofilaments in dendritic spines

Neurofilaments are one of the main cytoskeletal components in neurons and they can be found in the form of oligomers at pre- and postsynapses. How their presence is regulated at the postsynapse remains widely unclear. Here we systematically quantified by immunolabeling the occurrence of the neurofilament isoform triplet neurofilament light (NFL), medium (NFM), and heavy (NFH) at the postsynapse with STED nanoscopy together with markers of synaptic strength and activity. Our data shows that within dendritic spines neurofilament isoforms rarely colocalize with each other and that they are present to different extents, with NFL being the most abundant isoform. The amount of the three isoforms correlates with markers of postsynaptic strength and presynaptic activity to varying degrees: while NFL shows moderate correlation to both synaptic traits suggesting its involvement in synaptic response, NFM and NFH were only correlated on a low level. By quantifying the presence of neurofilaments at the postsynapse within the context of the synaptic status, this work sheds new light on the regulation of synaptic neurofilaments and their possible contribution to psychiatric disorders.

neuroscience↗

bioRxiv2021

A general design of caging-group free photoactivatable fluorophores for live-cell nanoscopy

The controlled switching of fluorophores between non-fluorescent and fluorescent states is central to every superresolution fluorescence microscopy (nanoscopy) technique, and the exploration of radically new switching mechanisms remains critical to boosting the performance of established, as well as emerging superresolution methods. Photoactivatable dyes offer significant improvements to many of these techniques, but often rely on photolabile protecting groups that limit their applications. Here we describe a general method to transform 3,6-diaminoxanthones into caging-group free photoactivatable fluorophores. These photoactivatable xanthones (PaX) assemble rapidly and cleanly into highly fluorescent, photo- and chemically stable pyronine dyes upon irradiation with light. The strategy is extendable to carbon- and silicon-bridged xanthone analogs, yielding a new family of photoactivatable labels spanning much of the visible spectrum. Our results demonstrate the versatility and utility of PaX dyes in fixed and live-cell fluorescence microscopy, and both coordinate-targeted stimulated emission depletion (STED) and coordinate-stochastic single-molecule localization superresolution microscopy (SMLM).

biochemistry↗

bioRxiv2021

Optimal Precision and Accuracy in 4Pi-STORM using Dynamic Spline PSF Models

Dual-objective 4Pi fluorescence detection enables single molecule localization microscopy, e.g. PALM and STORM, with sub-10 nanometer spatial resolution in 3D. Despite its outstanding sensitivity, wider application of this technique has been hindered by complex instrumentation requirements and the challenging nature of the data analysis. The point spread function (PSF) of the 4Pi optical system is difficult to model, leading to periodic image artifacts and compromised resolution. In this work we report the development of a 4Pi-STORM microscope which obtains improved resolution and accuracy by modeling the 4Pi PSF dynamically, while using a simpler optical design. We introduce dynamic spline PSF models, which incorporate fluctuations in the modulation phase of the experimentally determined PSF, capturing the temporal dynamics of the optical system. Our method reaches the theoretical limits for localization precision while largely eliminating phase-wrapping artifacts, by making full use of the information content of the data. With a 3D precision as high as 2 - 3 nanometers, 4Pi-STORM achieves new levels of image detail, and extends the range of biological questions that can be addressed by fluorescence nanoscopy, as we demonstrate by investigating protein and nucleic acid organization in primary neurons and mammalian mitochondria.

biophysics↗

bioRxiv2021

Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

cell biology↗

bioRxiv2020

Actin in Dendritic Spines Self-Organizes into a Critical State

It is known that dendritic spines change their size and shape spontaneously and sometimes to a large degree, but the function of this remains unclear. Here, we quantify these changes using time-series analysis of confocal data and demonstrate that spine size can follow different autoregressive integrated moving average (ARIMA) models and that shape- and size-changes are not correlated. We capture this behavior with a biophysical model, based on the spines actin dynamics, and find the presence of 1/f noise. When investigating its origins, the model predicts that actin in the dendritic spines self-organizes into a critical state, which creates a fine balance between static actin filaments and free monomers. We speculate that such a balance might be functionally beneficially to allow a spine to quickly reconfigure itself after LTP induction.

biophysics↗

bioRxiv2020

Overcoming efflux of fluorescent probes for actin imaging in living cells

Actin cytoskeleton is crucial for endocytosis, intracellular trafficking, cell shape maintenance and a wide range of other cellular functions. Recently introduced cell-permeable fluorescent actin probes suffer from poor membrane permeability and stain some cell populations inhomogeneously due to the active efflux by the plasma membrane pumps. We addressed this issue by constructing a series of probes which employ modified rhodamine fluorophores. We found that the best performing probes are based on 6-carboxy-carbopyronine scaffold. These probes show preferential binding to F-actin, do not require efflux pumps inhibitors for staining and can be used for 2D and 3D fluorescence nanoscopy at high nanomolar concentrations without significant cytotoxicity. We demonstrate their excellent performance in multiple organisms and cell types: human cell lines, frog erythrocytes, fruit fly tissues and primary neurons.

biochemistry↗

bioRxiv2020

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

physiology↗

bioRxiv2019

LUXendins reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and STED-compatible LUXendin651 we describe islet GLP1R expression patterns, reveal higher-order GLP1R organization including the existence of membrane nanodomains, and track single receptor subpopulations. We furthermore show that different fluorophores can confer agonistic behavior on the LUXendin backbone, with implications for the design of stabilized incretin-mimetics. Thus, our labeling probes possess divergent activation modes, allow visualization of endogenous GLP1R, and provide new insight into class B GPCR distribution and dynamics.

cell biology↗