Biology subjects

Brasselet, S.

Publications and source records attributed to Brasselet, S..

9 recordsLinked to original sources

bioRxiv2026

Nanoscale rheological heterogeneity revealed by Single Particle orientation Tracking (SPoT) of ultrashort carbon nanotubes in brain tissue

Transport in complex biological tissues is governed by local rheological heterogeneity at the nanoscale, yet probing such environments deep inside living systems remains challenging. Here, we introduce an orientation-sensitive single-particle tracking (SPoT) approach that simultaneously resolves translational and rotational dynamics of individual carbon nanotubes deep within biological tissue. By exploiting the intrinsic dipole-like emission and short-wave infrared luminescence of carbon nanotubes enhanced through the incorporation of quantum color-centers our method enables long-duration tracking with high signal-to-noise ratio in optically dense environments. Crucially, the length of these nanotubes can be precisely shortened down to a few tens of nanometers to adapt to diffusion environmental dimensions, further optimizing the tracking applicability. SPoT of single carbon nanotubes provides access to relative changes in local viscosity, steric constraints, and environmental anisotropy. When applied to the brain extracellular space, SPoT demonstrates that local variations in the translational and rotational diffusion of tracers are heterogeneous and not systematically correlated. By combining trajectory-based signatures of confinement with rotational dynamics, this analysis allows the local contributions of effective viscosity and spatial tortuosity to be disentangled. By enabling combined translational and rotational tracking of nano-emitters over unprecedented depths and timescales, this work establishes a new framework for probing nanoscale transport and rheological heterogeneity in intact biological tissues and more generally in complex diffusive environments.

biophysics↗

bioRxiv2025

SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion.

When stimulated by nucleation-promoting factors such as WAVE, the Arp2/3 complex generates branched actin networks. In contrast, when activated by SPIN90, the Arp2/3 complex generates linear actin filaments. The Arp2/3 complex in mammals, consists of 8 iso-complexes with dieerent properties as there are two isoforms of Arp3, ArpC1 and ArpC5. Here, using recombinant Arp2/3 iso-complexes with defined compositions, we show that SPIN90 selectively activates ArpC5L- rather than ArpC5- containing complexes to generate linear actin filaments. Consistent with this, SPIN90 at the leading edge of migrating cells enhances the recruitment of ArpC5L-, but not ArpC5- containing, Arp2/3 complexes to lamellipodia. These SPIN90-Arp2/3-ArpC5L complexes generate linear actin filaments that integrate into lamellipodia and impact protrusion eeiciency. Moreover, using polarised light microscopy, we show that loss of SPIN90 leads to an enrichment in actin filaments oriented more perpendicular to the plasma membrane. Our results demonstrate that SPIN90 regulates the architecture and dynamics of lamellipodial actin in an Arp2/3 iso-complex dependent fashion. SummarySPIN90 selectively recruits and activates ArpC5L containing Arp2/3 to shape the architecture and dynamics of lamellipodial actin.

cell biology↗

bioRxiv2025

SMOLM-LFM: ratiometric single molecule orientation without polarizers

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

bioengineering↗

bioRxiv2025

Polarization MultiFocus Microscopy for volumetric super-resolution and orientation imaging of biofilaments

Accessing molecular orientation in single molecule localization microscopy (SMLM) offers valuable insights into molecular ordering and organization in biological structures. Conventional single-molecule orientation-localization microscopy (SMOLM) methods typically rely on either engineering the microscopes point-spread function (PSF) to encode the orientation information or on polarization resolved detection. While PSF engineering enables detailed orientation analysis, it often requires complex computational analysis and suffers from reduced performance in dense cellular environments due to PSF spreading and overlap. In contrast, polarization-based approaches are easier to implement and are more fit when imaging dense samples but are unable to retrieve the axial information of single molecules. To overcome this limitation, we introduce the Polarization MultiFocus Microscope (PolMFM), a novel method for simultaneously retrieving the orientation and 3D position of single molecules. PolMFM combines the orientation measurement capabilities of a 4-polarization splitting scheme with a 3-planes multifocus microscope (MFM) enabling the reconstruction of molecular 2D orientation, wobble, and axial localization in a single acquisition. Through simulations, we demonstrate that PolMFM accurately recovers both orientation and 3D position, despite PSF defocusing. Experimental validation with reference samples shows that PolMFM matches the orientation precision of 4-Polar STORM, while uniquely adding axial information. We demonstrate the power of PolMFM by resolving the orientation and 3D positions of molecules in actin filaments in fixed cells, and by revealing that chromatin in crickets undergoes major reorganization and increased ordering during spermiogenesis. These findings highlight the potential of PolMFM for high-precision, multidimensional super-resolution imaging in complex and crowded biological environments.

biophysics↗

bioRxiv2025

4polar3D : Single molecule 3D orientation imaging of dense actin networks using ratiometric polarization splitting

Single Molecule Orientation and Localization Microscopy (SMOLM) aims at simultaneously measuring the position and orientation of single molecules, generating orientation-encoded super-resolved images by estimating both their 3D mean orientation and the extent of their angular fluctuations (wobble). Most existing SMOLM approaches rely on the engineering of single molecules point spread functions, which requires complex optical setups and long computational times that can be an obstacle in dense cellular environments with high detection density and challenging imaging conditions. In this work, we propose a simpler and effective method named 4polar3D, based on the estimation of single molecule intensities projected onto four polarized channels with controlled numerical apertures. This strategy enables 3D orientation measurements of single molecules in addition to their 2D localization in a fast processing step. We demonstrate that 4polar3D can resolve nanoscale molecular organization in whole cells crowded structures, uncovering 3D-oriented actin filament networks in densely packed lamellipodia and podosomes.

bioengineering↗

bioRxiv2024

Genetically encoded reporters of actin filament organization in living cells and tissues

The cytoskeletal protein actin is crucial for cell shape and integrity throughout eukaryotes. Actin filaments perform essential biological functions, including muscle contraction, cell division and tissue morphogenesis. These diverse activities are achieved through the ability of actin filaments to be arranged into precise architectures. Much progress has been made in defining the proteome of the actin cytoskeleton, but a detailed appreciation of the dynamic organizational state of the actin filaments themselves has been hindered by available tools. Fluorescence polarization microscopy is uniquely placed for measuring actin filament organization by exploiting the sensitivity of polarized light excitation to the orientation of fluorophores attached to actin filaments. By engineering fusions of five widely used actin localization reporters to fluorescent proteins with constrained mobility, we have succeeded in developing genetically-encoded, green- and red-fluorescent-protein-based reporters for non-invasive, quantitative measurements of actin filament organization in living cells and tissues by fluorescence polarization microscopy.

cell biology↗

bioRxiv2022

Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

cell biology↗

bioRxiv2021

4polar-STORM polarized super-resolution imaging of actin filament organization in cells

Advances in single-molecule localization microscopy are providing unprecedented insights into the nanometer-scale organization of protein assemblies in cells and thus a powerful means for interrogating biological function. However, localization imaging alone does not contain information on protein conformation and orientation, which constitute additional key signatures of protein function. Here, we present a new microscopy method which combines for the first time Stochastic Optical Reconstruction Microscopy (STORM) super-resolution imaging with single molecule orientation and wobbling measurements using a four polarization-resolved image splitting scheme. This new method, called 4polar-STORM, allows us to determine both single molecule localization and orientation in 2D and to infer their 3D orientation, and is compatible with high labelling densities and thus ideally placed for the determination of the organization of dense protein assemblies in cells. We demonstrate the potential of this new method by studying the nanometer-scale organization of dense actin filament assemblies driving cell adhesion and motility, and reveal bimodal distributions of actin filament orientations in the lamellipodium, which were previously only observed in electron microscopy studies. 4polar-STORM is fully compatible with 3D localization schemes and amenable to live-cell observations, and thus promises to provide new functional readouts by enabling nanometer-scale studies of orientational dynamics in a cellular context.

biophysics↗

bioRxiv2017

Lipid order degradation in autoimmune demyelination probed by polarization resolved coherent Raman microscopy

Myelin around axons is currently widely studied by structural analyses and large scale imaging techniques, with the goal to decipher its critical role in neuronal protection. While there is strong evidence that in myelin, lipid composition and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify molecular order of lipids in myelin at sub-diffraction scales, using label-free polarization resolved Coherent Anti Stokes Raman (PR-CARS), which exploits CARS sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits to unravel molecular-scale perturbations of lipid layers at early stage of the demyelination progression, while the membrane architecture at the mesoscopic scale (here about 100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and opens new prospectives towards molecular-scale understanding of neurodegenerative diseases.

biophysics↗