Biology subjects

Szalai, A. M.

Publications and source records attributed to Szalai, A. M..

8 recordsLinked to original sources

bioRxiv2026

Tau isoforms modulate the axon initial segment controlling axonal trafficking and neuronal excitability

The axon initial segment (AIS) is a specialized neuronal compartment integrating action potential initiation with selective control of axonal trafficking. The microtubule associated protein tau is a central regulator of cytoskeletal organization and transport, yet how distinct tau isoforms contribute to AIS development and function remains unclear. Here, we examined the role of tau isoform relative abundance in regulating AIS establishment, maturation, excitability, and transport selectivity using murine primary neurons and human induced pluripotent stem cell (hiPSC)-derived neurons combined with super resolution imaging, electrophysiology, and live trafficking assays. We found that tau expression levels and isoform content modulate the timing and robustness of AIS maturation. In murine neurons, tau deficiency or predominance of 3 repeat (3R) tau delays Ankyrin G accumulation and AIS stabilization without preventing AIS formation. hiPSC-derived neurons display an intrinsic AIS developmental program accompanied by progressive changes in tau isoform content. Super resolution DNA PAINT reveals that endogenous tau decorates axonal microtubules in discrete nanoclusters with compartment specific distributions. Modulation of the endogenous 3R/4R tau balance in hiPSC-derived neurons shows that isoform composition, independently of tau levels, regulates AIS positioning and Ankyrin G organization. Functionally, shifts towards 3R-tau reduce sodium currents, impair action potential firing, and alter lysosomal transport dynamics within the AIS. Together, these findings identify tau isoform balance as a developmental regulator of AIS maturation, linking cytoskeletal organization to neuronal excitability and transport gating. Because the aberrant alternative tau splicing of exon 10 is a defining feature of primary tauopathies, our results provide mechanistic insight into how imbalanced tau isoforms may contribute to neuronal dysfunction.

cell biology↗

bioRxiv2026

Resolving Sub-Microsecond Conformational Dynamics of Vertical Nucleic Acids on Graphene

The function of nucleic acids is governed not only by their structure but also by their dynamics. At the molecular scale, transitions between functional structural states are superimposed on rapid thermal fluctuations, resulting in an intricate interplay that is challenging to resolve experimentally, particularly at the single-molecule level. Here, we introduce a novel approach for unraveling sub-microsecond dynamics in oligonucleotides, enabling direct observation of fluctuations in single DNA molecules. By immobilizing nucleic acids vertically on graphene and exploiting distance-dependent graphene energy transfer of fluorescent molecules attached to the DNA, we relate fluctuations in fluorescence intensity to biomolecular dynamics. We show that ionic strength modulates the fluctuations and that structural defects in DNA, such as nucleotide gaps or mismatches, alter the measured dynamics. The experimental findings are complemented by atomistic molecular dynamics simulations and kinetic Monte Carlo simulations, establishing a direct link between theoretical predictions of structure and dynamics and experimentally accessible fluctuation timescales. Overall, our findings advance the understanding of how thermal fluctuations affect oligonucleotides and are modulated by both external and internal stimuli.

biophysics↗

bioRxiv2026

Distinct nanoscale membrane organizations of mucins and trans-sialidases in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, relies on surface sialylation to evade host immunity and invade cells. This process is mediated by trans-sialidases (TS) and mucins, an enzyme-substrate pair anchored to distinct lipid environments. Yet, how these molecules are organized at the nanoscale in the parasites membrane remains unknown. Using dual-color super-resolution microscopy, we show that [~]60% of mucins and TS are segregated into nanoclusters ([~]100 nm) that rarely contact each other, distributed with a non-random separation distance, indicating that these abundant domains follow a specific order in the plasma membrane and are unlikely to serve as primary sites of sialylation. In contrast, the [~]40% fraction of non-clustered mucins and TS exhibits significantly shorter-than-random separation distances and appear ordered as in a shared fibrillar network. Additionally, we describe a distinct structural organization within these domains: mucins --residents of detergent-resistant domains (DRDs)--organize into high-molecular-weight complexes, whereas TS (which are excluded from DRDs) do not. This reveals an additional layer of membrane asymmetry and suggests a potential mechanism for domain-specific protein localization. Together, these findings uncover major principles of T. cruzi surface organization, with important implications for the regulation of host-parasite interactions.

microbiology↗

bioRxiv2026

Nanoscale organization of betaII-spectrin within segments of the membrane-associated periodic skeleton in mouse sciatic nerve axons

The actin/spectrin membrane-associated periodic skeleton (MPS) is a ubiquitous cytoskeletal structure essential for axonal integrity and function. Primarily studied in cultured neurons, the MPS has been extensively modeled as actin rings spaced by spectrin tetramers, the latter assumed to be regularly and densely distributed across the axonal perimeter. However, its nanoscale organization within native tissue environments remains poorly understood. In this study, we investigated the three-dimensional organization of {beta}II-spectrin in the mouse sciatic nerve using 3D-dSTORM and STED super-resolution microscopy on thin transversal cryosections. By implementing a custom quantitative analysis pipeline, we resolved the sub-diffraction architecture of the MPS across myelinated axons of diverse diameters. We confirm that {beta}II-spectrin is localized to the inner face of the axonal plasma membrane and maintains a longitudinal periodicity of approximately 170 nm, consistent with previous observations. Crucially, 3D-dSTORM revealed that {beta}II-spectrin along the axonal perimeter is organized in discrete nanoscale clusters with a median effective radius of 25 nm, compatible with the size of an individual spectrin tetramer visualized by indirect immunolabeling. The number of these clusters scales linearly with the axonal perimeter, maintaining a constant membrane occupancy of [~]20% across varying axon diameters. Moreover, these clusters exhibit a non-random spatial distribution with a characteristic center-to-center nearest-neighbor distance of [~]200 nm. These findings challenge simplified models of the MPS based on cultured systems and demonstrate that the MPS in peripheral nerves is composed of discrete structural units. This modular, dispersed organization may provide the structural flexibility required to withstand the mechanical demands of the peripheral nervous system while maintaining a stable periodic scaffold.

neuroscience↗

bioRxiv2025

Minimally Invasive DNA-Mediated Photostabilization for Extended Single-Molecule and Super-resolution Imaging

Photobleaching of fluorescence labels poses a major limitation in single-molecule and super-resolution microscopy. Conventional photostabilization methods, such as oxygen removal and addition of high concentrations of photostabilization additives, often require careful fluorophore selection and can disrupt the biological environment. To address these limitations, we developed a modular and minimally invasive photostabilization approach that utilizes DNA-mediated delivery of a photostabilizer directly to the imaging site. Under lower excitation intensities, the DNA-mediated strategy outperformed solution-based approaches, achieving efficient photostabilization at significantly lower additive concentrations. However, at higher excitation intensities, the stability of a single photostabilizer molecule became the limiting factor. To overcome this and reduce the loss of localizations in DNA-PAINT experiments we have also implemented a recovery scheme where the photostabilizer is continuously replenished at the imaging site. We further extended the approach to cell imaging, demonstrating improved localization rate and precision in 3D-DNA PAINT measurements. DNA-mediated photostabilization offers a promising solution for imaging applications where high additive concentrations are prohibited. Its modularity enables adaptation to various imaging schemes and ultimately expands the repertoire of fluorophores suitable for single-molecule and super-resolution imaging.

biophysics↗

bioRxiv2023

Real-Time Structural Biology of DNA and DNA-Protein Complexes on an Optical Microscope

The intricate interplay between DNA and proteins is key for biological functions such as DNA replication, transcription, and repair. To better understand these interactions, it is crucial to develop tools to study DNA-protein complexes with high spatial and temporal resolution. Here, we use the vertical orientation that DNA adopts on graphene and investigate its interactions with proteins via energy transfer from a probe dye to graphene, achieving spatial resolution down to the [A]ngstrom scale. We measured the bending angle of DNA induced by adenine tracts, bulges, abasic sites and the binding of Escherichia coli endonuclease IV with unprecedented precision and millisecond time resolution. Additionally, we observed the translocation of the O6-alkylguanine DNA alkyltransferase along double-stranded DNA, reaching single-base pair resolution and detecting an affinity for adenine tracts. Overall, we foresee that this method will become a widespread tool for the dynamical study of nucleic acid and nucleic acid-protein interactions.

biophysics↗

bioRxiv2023

Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localizes in growth cones of undifferentiated neurites, while in developing axons it displays a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevents axon initiation but has no effect on elongation, while formin inhibition reduces axon extension without significantly altering initial outgrowth. Besides, RhoA-mDia promotes axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK that restrains growth cone microtubule assembly and protrusion. Finally, we show that similar mechanisms might operate during axonal regeneration, with RhoA-ROCK slowing axon regrowth after axotomy and RhoA-mDia favoring extension of regenerated axons.

neuroscience↗

bioRxiv2019

Three-dimensional total internal reflection fluorescence nanoscopy with sub-10 nm resolution

Single-molecule localization microscopy (SMLM) enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single molecules image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present SIMPLER, a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence (TIRF) microscope. SIMPLER requires no hardware modification whatsoever to a conventional TIRF microscope, and complements any 2D SMLM method to deliver 3D images with nearly isotropic nanometric resolution. Examples of the performance of SIMPLER include the visualization of the nuclear pore complex through dSTORM with sub-20 nm resolution and of microtubule cross-sections resolved with sub-10 nm through DNA-PAINT.

biophysics↗