Biology subjects

Loza-Alvarez, P.

Publications and source records attributed to Loza-Alvarez, P..

8 recordsLinked to original sources

bioRxiv2026

Single-disc optical visualization in photoreceptors uncovers protein architecture and compartmentalized pathology

Photoreceptor discs are densely stacked membranes housing visual pigments, yet their tight 35 nm spacing has kept molecular organization beyond the reach of optical microscopy. Here, we employ iterative ultrastructure expansion microscopy (iU-ExM) to achieve the first optical visualization of proteins within individual photoreceptor discs at 12 nm effective resolution through 20-fold expansion. We demonstrate that rhodopsin occupies 92% of the disc radial extent in situ, substantially exceeding the [~]50% area fraction reported from atomic force microscopy of isolated membranes. We provide the first optical detection of peripherin-2 within disc incisures and resolve connecting cilium and centriolar appendage architecture in three dimensions. Application to the RCS rat model of retinitis pigmentosa reveals compartmentalized pathology: outer segment disc spacing increased 29% while centriolar architecture remained preserved, suggesting intact protein trafficking during early degeneration. By bridging molecular identification with ultrastructural context, this work opens optical access to individual membrane compartments within densely packed cellular architectures previously resolved only by electron microscopy.

cell biology↗

bioRxiv2026

PSF-Driven Spatio-Temporal Blending in Fluorescence Lifetime Imaging Microscopy and Its Mitigation via Mean-Shift Super-Resolution-Based Masking.

Fluorescence Lifetime Imaging Microscopy (FLIM) enables quantitative mapping of molecular environments in living systems with high biochemical specificity. However, spatial overlap dictated by the diffraction-limited point spread function (PSF) causes a mixing of temporal signals: photons from neighboring emitters collected within the same pixel yield composite decay profiles, generating apparent intermediate lifetimes that can be mistaken for variations in the local molecular environment. We introduce a workflow that applies Mean-Shift Super-Resolution (MSSR) to raw intensity data to generate intensity-derived spatial masks prior to phasor-based lifetime analysis. The method is computationally efficient and preserves decay kinetics because it operates on intensity-derived spatial information rather than modifying temporal data. In U2OS cells labeled with spectrally-overlapping fluorophores, phasor analysis reveals an intermediate lifetime population localized at PSF-overlap interfaces, consistent with optical mixing rather than intrinsic lifetime heterogeneity. MSSR-derived masking suppressed this mixed population while preserving stable phasor cluster centers -i.e. the distribution of similar phasor coordinates in the phasor plane- for each fluorophore. Simulations of strictly monoexponential fluorescence decay emitters further show that blended lifetime decay profiles are present at separations up to 4{sigma} and becomes maximal near [~]1.6{sigma}, indicating that conventional spatial resolution criteria can underestimate lifetime cross-talk. Application of this workflow to three-component FLIM showed also a reduced overlap of pixel distributions in phasor plots while maintaining distinct lifetime signatures. Overall, MSSR-based spatial refinement provides an accessible strategy to improve the spatial resolution while maintaining accuracy of FLIM measurements.

biophysics↗

bioRxiv2024

Neuro-immunology in a mouse model of anti-NMDAR encephalitis and assessment of treatment approaches

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a disorder mediated by autoantibodies against the GluN1 subunit of NMDAR. It occurs with severe neuropsychiatric symptoms that often improve with immunotherapy. Clinical studies and animal models based on patients antibody transfer or NMDAR immunization suggest that the autoantibodies play a major pathogenic role. Yet, there is an important need of models offering an all-inclusive neuro-immunobiology of the disease together with a clinical course long enough to facilitate the assessment of potential new treatments. Toward this end, eight-week-old female mice (C57BL/6J) were immunized (days 1 and 28) with GluN1356-385 peptide or saline with AddaVax adjuvant and pertussis toxin. After symptom development ([~]day 35), subsets of mice were treated with an anti-CD20 (day 35), a positive allosteric modulator (PAM) of NMDAR (NMDAR-PAM, SGE-301) from days 45 to 71, or both. GluN1-antibody synthesis, epitope spreading, effects of antibodies on density and function of NMDAR, brain immunological infiltrates, microglial activation and NMDAR phagocytosis, and antibody synthesis in cultured inguinal and deep cervical lymph nodes (DCLN) were assessed with techniques including immunohistochemistry, calcium imaging, confocal and super-resolution microscopy, electrophysiology, or flow cytometry. Changes of memory and behaviour were assessed with a panel of behavioural tests, and clinical/subclinical seizures with brain-implanted electrodes. Immunized mice, but not controls, developed serum and CSF NMDAR-antibodies (IgG1 predominant) against the immunizing peptide and other GluN1 regions (epitope spreading) resulting in a decrease of synaptic and extrasynaptic NMDAR clusters and reduction of hippocampal plasticity. These findings were associated with brain inflammatory infiltrates, mainly B- and plasma cells, microglial activation, colocalization of NMDAR-IgG complexes with microglia, and presence of these complexes within microglial endosomes. Cultures of DCLC showed GluN1-antibody production. These findings were associated with psychotic-like behaviour (predominant at disease onset), memory deficit, depressive-like behaviour, abnormal movements (15% of mice), and lower threshold for developing pentylenetetrazole-induced seizures (hypoactivity, myoclonic jerks, continuous tonic-clonic) which correlated with regional cFOS expression. Most symptoms and neurobiological alterations were reversed by the anti-CD20 and PAM, alone or combined. Initial repopulation of B cells, by the end of the study, was associated with re-emergence of clinical-neurobiological alterations, which were abrogated by PAM. Overall, this model offers an all-inclusive neuro-immunobiology of the disease, allowing testing novel treatments, supporting the potential therapeutic role of NMDAR-PAM, and suggesting an immunological paradigm of systemic antigen presentation and brain NMDAR epitope spreading, which along the DCLN might contribute to fine-tune the polyclonal immune response.

neuroscience↗

bioRxiv2023

Dynamics of macrophage polarization support Salmonella persistence in a whole living organism

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high-resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages confirmed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

immunology↗

bioRxiv2022

Volumetric bioluminescence imaging of cellular dynamics with deep learning based light-field reconstruction

The application of genetically encoded fluorophores for microscopy has afforded one of the biggest revolutions in the biosciences. Bioluminescence microscopy is an appealing alternative to fluorescence microscopy, because it does not depend on external illumination, and consequently does neither produce spurious background autofluorescence, nor perturb intrinsically photosensitive processes in living cells and animals. The low quantum yield of known luciferases, however, limit the acquisition of high signal-noise images of fast biological dynamics. To increase the versatility of bioluminescence microscopy, we present an improved low-light microscope in combination with deep learning methods to increase the signal to noise ratio in extremely photon-starved samples at millisecond exposures for timelapse and volumetric imaging. We apply our method to image subcellular dynamics in mouse embryonic stem cells, the epithelial morphology during zebrafish development, and DAF-16 FoxO transcription factor shuttling from the cytoplasm to the nucleus under external stress. Finally, we concatenate neural networks for denoising and light-field deconvolution to resolve intracellular calcium dynamics in three dimensions of freely moving Caenorhabditis elegans with millisecond exposure times. This technology is cost-effective and has the potential to replace standard optical microscopy where external illumination is prohibitive.

bioengineering↗

bioRxiv2021

Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we used zebrafish to further understand the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was panneuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

neuroscience↗

bioRxiv2020

The microtubule organization in the zebrafish yolk adapts to transgene-mediated phenotypic variations

The organization of microtubule networks in the cells is orchestrated by subcellular structures named microtubule organizing centers (MTOCs). In zebrafish embryos, the yolk is surrounded by a cytoplasmic layer containing a vast network of microtubules. In order to understand how this complex network is organized, we use dclk2-GFP zebrafish embryos, as a microtubule reporter line, and Light Sheet Fluorescence Microscopy. We find that this organization is mediated by a variable number of aster-like MTOCs during epiboly, and that it does not follow a rigid scheme, exemplifying developmental robustness. We characterize asters morphology, dynamics, and their uniform distribution in the yolk sphere. Consistent with their role as MTOCs we find that they contain key molecular machinery for MTs dynamics, amongst which centrin marks the assignation of MTOCs over time. Finally, we demonstrate that merely the overexpression of dclk2-GFP in wild type embryos can induce the formation of asters. We propose dclk2-GFP embryos as a model for the study of the collective behaviour of microtubules in complex systems.

developmental biology↗

bioRxiv2019

The nucleus measures shape deformation for cellular proprioception and regulates adaptive morphodynamics

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within their local environment remains largely unknown. Here we show that the nucleus, the biggest cellular organelle, functions as a non-dissipative cellular shape deformation gauge that enables cells to continuously measure shape variations on the time scale of seconds. Inner nuclear membrane unfolding together with the relative spatial intracellular positioning of the nucleus provides physical information on the amplitude and type of cellular shape deformation. This adaptively activates a calcium-dependent mechano-transduction pathway, controlling the level of actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behaviour to their microenvironment. One Sentence SummaryThe nucleus functions as an active deformation sensor that enables cells to adapt their behavior to the tissue microenvironment.

cell biology↗