Biology subjects

do Rego Barros Fernandes Lima, M. A.

Publications and source records attributed to do Rego Barros Fernandes Lima, M. A..

4 recordsLinked to original sources

bioRxiv2026

Single-stranded telomeric repeats segregate into spatial compartments within ALT-associated PML bodies

Alternative lengthening of telomeres (ALT)-associated PML bodies (APBs) concentrate single-stranded (ss) telomeric DNA and RNA species that are critical for recombination-based telomere maintenance. However, how these species are organized inside APBs has remained invisible at microscopic resolution. Here, we map the nanoscale topology of APB components using 3D MINFLUX super-resolution microscopy combined with multiplexed exchange DNA-PAINT labeling at [~]3 nm localization precision. We discover that ssC-rich and ssG-rich telomeric repeats occupy distinct spatial compartments within a partially open, [~]70 nm thick PML protein shell: ssC-rich repeats concentrate at the inner shell surface, while ssG-rich repeats distribute broadly through the interior alongside TRF1-marked double-stranded telomeric chromatin. The ssG-rich signal is predominantly DNA and frequently colocalizes with POT1 assemblies. Additional TRF1 clusters outside the shell indicate multi-telomere association. Together, these structural constraints motivate a model of ALT in which t-loop resolution generates a C-circle template that drives rolling-circle amplification of telomeric repeats. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/724127v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@fdb6a2org.highwire.dtl.DTLVardef@e185c9org.highwire.dtl.DTLVardef@168b12org.highwire.dtl.DTLVardef@44bd0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI3D MINFLUX combined with exchange DNA-PAINT resolves APB components at {bsim}3 nm isotropic precision, enabling direct visualization of molecular organization beyond the reach of conventional super-resolution microscopy. C_LIO_LIPML forms a partially open, [~]70 nm thick spherical shell in APBs with reduced molecular density compared to canonical PML nuclear bodies, providing the structural scaffold and coordinate system for mapping internal APB organization. C_LIO_LIDouble-stranded telomeric chromatin (TRF1-marked) fills the APB interior, with additional TRF1 domains outside the shell indicative of multi-telomere clustering at individual APBs. C_LIO_LIssC-rich repeats concentrate in the inner part of the PML shell while ssG-rich DNA repeats distribute broadly through the interior, revealing strand-specific spatial segregation. C_LIO_LIThe ssG-rich single-stranded repeats in APBs predominantly comprise DNA and partially colocalize with POT1, connecting their spatial distribution to single-stranded telomeric DNA substrates. C_LIO_LIThe spatial organization of APB components motivates a mechanistic model in which t-loop processing generates C-circle templates for rolling-circle amplification, while multi-telomere clustering facilitates inter-telomeric recombination. C_LI

cell 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↗

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↗

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↗