Biology subjects

Almeida, A. C.

Publications and source records attributed to Almeida, A. C..

6 recordsLinked to original sources

bioRxiv2025

Tubulin autoregulation tunes microtubule dynamics to support multicellular architecture and viability

Alpha- and beta-tubulin heterodimers dynamically assemble into microtubules, key cytoskeletal elements involved in intracellular trafficking, cell adhesion and division. The availability of free tubulins regulates the synthesis of new subunits. In response to excessive soluble {beta}-tubulins, tetratricopeptide protein 5 (TTC5) selectively recognizes nascent tubulins at the ribosome, recruiting downstream effectors that degrade their encoding messenger RNAs, in a process known as tubulin autoregulation. Despite its well-characterized molecular framework, the biological relevance of this regulatory pathway remains unknown. Here, using human 3D cellular models, advanced optics, and genetic perturbation of tubulin biosynthesis, we reveal that loss of TTC5-dependent tubulin autoregulation elevates soluble tubulin levels, inducing microtubule hyperstability, and disrupting cytoskeletal organization. These defects impair the localization of adhesion molecules at cell-cell junctions and extracellular matrix interfaces, compromising tissue architecture and reducing overall cell viability. Our findings establish tubulin autoregulation as a critical mechanism that tunes microtubule dynamics to sustain cellular integrity and tissue homeostasis.

cell biology↗

bioRxiv2024

Soluble αβ-tubulins reversibly sequester TTC5 to regulate tubulin mRNA decay

Microtubules, built from heterodimers of - and {beta}-tubulins, control cell shape, mediate intracellular transport and power cell division. The concentration of {beta}-tubulins is tightly controlled through a post-transcriptional mechanism involving selective and regulated degradation of tubulin-encoding mRNAs. Degradation is initiated by TTC5, which recognizes tubulin-synthesizing ribosomes and recruits downstream effectors to trigger mRNA deadenylation. Here, we have investigated how cells regulate TTC5 activity. Biochemical and structural proteomic approaches reveal that under normal conditions, soluble {beta}-tubulins bind to and sequester TTC5, preventing it from engaging nascent tubulins at translating ribosomes. We identify the flexible C-terminal tail of TTC5 as a molecular switch, toggling between soluble {beta}-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble {beta}-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

cell biology↗

bioRxiv2023

Development of a SARS-COV-2 monoclonal antibody panel and its applicability as a reagent in high-throughput fluorescence reduction neutralization and immunohistochemistry assays

Since its emergence in late 2019, infection by SARS-CoV-2 (COVID-19 disease) has quickly spread worldwide, leading to a pandemic that has caused millions of deaths and huge socio-economic losses. Although vaccination against COVID-19 has significantly reduced disease mortality, it has been shown that protection wanes over time, and that circulating SARS-CoV-2 variants may escape vaccine-derived immunity. Therefore, serological studies are still necessary to assess protection in the population and better guide vaccine booster programs. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard method for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. In this study, we developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we also developed and characterized in-house monoclonal antibodies (mAbs) to lower assay costs and reduce the vulnerability of the test to reagent shortages. Using samples collected before the pandemic and from individuals vaccinated against COVID-19, we showed that the results of the FRNA we developed using commercial and in-house mAbs strongly correlated with those of the standard PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess other SARS-CoV-2 variants of concern (VOCs).

microbiology↗

bioRxiv2021

A mitotic screen in Indian muntjac cells unveils how Augmin drives kinetochore fiber maturation

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fibers. How k-fibers mature from initial kinetochore-microtubule attachments remains a fundamental question. By combining functional perturbations in Indian muntjac, a placental mammal with the lowest known chromosome number (n=3) and distinctively large kinetochores, with fixed- and subsecond live-cell super-resolution CH-STED nanoscopy, we uncovered the mechanism by which Augmin mediates k-fiber maturation. Augmin promoted kinetochore microtubule turnover by sustaining microtubule formation from kinetochores and poleward flux, regardless of pioneer centrosomal microtubules. Tracking of microtubule growth events within individual k-fibers revealed a wide angular dispersion, consistent with Augmin-mediated branched microtubule nucleation. Augmin depletion reduced the frequency of microtubule growth events within k-fibers and hampered normal repair after acute k-fiber injury by laser microsurgery. Our work underscores the contribution of microtubule formation from kinetochores and Augmin-mediated microtubule amplification for k-fiber maturation and spindle assembly in mammals.

cell biology↗

bioRxiv2019

Malassezia interaction with a reconstructed human epidermis: imaging studies

BackgroundBiofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. ObjectiveThe present study aimed to evaluate Malassezia interaction with a reconstructed human epidermis (RhE) model. MethodsMalassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrheic dermatitis. Yeasts of two strains of M. furfur and M. sympodialis were inoculated onto the SkinEthic RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning-electron microscopy. ResultsColonization of the RhE surface with aggregates of Malassezia yeasts entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeasts were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded (not seen in control samples). Slight differences were found in the biofilm architectural structure between the two tested species. ConclusionSkin isolates of M. furfur and M. sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found with higher entrapment by a denser and more gelatinous extracellular matrix in M. furfur biofilm.

microbiology↗

bioRxiv2018

Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally breaks the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

biophysics↗