Biology subjects

Stefani, F. D.

Publications and source records attributed to Stefani, F. D..

7 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

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↗

bioRxiv2024

Direct single-molecule detection and super-resolution imaging with a low-cost portable smartphone-based microscope

We present a novel, low-cost, portable smartphone-based fluorescence microscope capable of directly detecting single molecules without signal amplification. The setup leverages the image sensors and data handling capacity of mass-produced smartphones, making it adaptable to any smartphone and capable of detecting single molecules across the visible spectral range. We showcase this capability through single-molecule measurements on DNA origami models and super-resolution microscopy of biological cells by single-molecule localization microscopy. This development paves the way for biotechnology innovations making use of massively distributed or personalized assays with single-molecule sensitivity with the potential to revolutionize digital bioassays, point-of-care diagnostics, field expeditions, STEM outreach, and life science education.

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↗

bioRxiv2023

Super-Resolved FRET and Co-Tracking in pMINFLUX

Single-molecule FRET (smFRET) is widely used to investigate dynamic (bio) molecular interactions taking place over distances of up to 10 nm. With the advent of recent super-resolution methods such as MINFLUX, MINSTED or RASTMIN, the spatiotemporal resolution of these techniques advanced towards the smFRET regime. While these methods do not suffer from the spatial restriction of FRET, they only visualize one emitter at a time, thus rendering fast dynamics of interactions out of reach. Here, we describe two approaches to overcome this limitation in pMINFLUX using its intrinsic fluorescence lifetime information. First, we combined pMINFLUX with smFRET. This enabled us to track a FRET donor fluorophore and simultaneously colocalize its FRET acceptor with nanometer precision. To extend co-localized tracking beyond the FRET range, we developed pMINFLUX lifetime multiplexing, a method to simultaneously track two fluorophores with similar spectral properties but distinct fluorescence lifetimes. We demonstrated its application on both static and dynamic DNA origami systems with a precision better than 2 nm. Within the FRET range, pMINFLUX lifetime multiplexing additionally uses a novel combined phasor-microtimegating approach. This paves the way for nanometer precise co-localized tracking for inter-dye distances between 4 nm and 100 nm, closing the resolution gap between smFRET and co-tracking. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/534096v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@11459forg.highwire.dtl.DTLVardef@1f1cee2org.highwire.dtl.DTLVardef@4592e6org.highwire.dtl.DTLVardef@1af3e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

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↗