Biology subjects

dos Santos, A.

Publications and source records attributed to dos Santos, A..

14 recordsLinked to original sources

bioRxiv2025

Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

Proteasomes are proteolytic machines essential for proteostasis and genome integrity. They consist of a 20S core particle capped by regulatory complexes that confer substrate selectivity (Voges, 1999). Despite evidence that proteasome function is necessary for successful spermatogenesis and fertilisation (Sutovsky, 2011; Xiong et al., 2022), the presence, composition, and role of proteasomes within the sperm nucleus have remained a subject of debate. Here, we used in situ electron cryo-tomography (cryo-ET) in human sperm cells to elucidate the molecular architecture of nuclear proteasomes, which cluster in DNA-free, nuclear cavities within the sperm nucleus. Subtomogram averaging revealed that the main population of proteasomes consists of 20S core particles, with a smaller fraction of 20S capped by PA200 activator, solved at sub-nanometre resolution. Subsequent single-particle cryo-EM analysis of purified native human sperm proteasomes further elucidated atomic features of the essential testis-specific subunit 4s, showing the presence of a unique splice variant. Notably, our reconstruction resolved a native peptide in the catalytic {beta}2 subunit, providing novel insights into the proteolysis mechanism and PA200-mediated enhancement of trypsin activity. Finally, we show nuclear enrichment of proteasomes during sperm cell differentiation in human testis tissue, with 20S and PA200 clustering following meiosis, at the spermatid stage. Our findings shed light on the unique organisation and compositional diversity of nuclear proteasomes in human sperm cells, disclosing novel molecular insights into their catalytic function.

cell biology↗

bioRxiv2025

Myosin VI orchestrates estrogen-driven gene expression in breast cancer cells.

Approximately 75% of breast cancer cases are estrogen receptor (ER)-positive, with endocrine therapies forming the foundation of treatment. However, therapeutic resistance remains a major clinical challenge, necessitating the identification of new molecular targets. Myosin VI (MVI), a motor protein increasingly linked to cancer, directly interacts with the ER and plays a key role in the spatial regulation of RNA Polymerase II. Notably, expression levels of MVI and ER are positively correlated across breast cancer tissues. Here, we present a multidisciplinary investigation combining advanced imaging, genomic profiling, and phenotypic characterisation to elucidate the interplay between MVI and the ER. We demonstrate that MVI nuclear localisation is dynamically regulated by estrogen signalling and ER activity. Conversely, ER nuclear localisation requires active MVI, suggesting a reciprocal regulatory mechanism. Furthermore, MVI influences subnuclear architecture, modulating ER transcriptional activity and downstream gene expression programmes that govern cell proliferation and migration. Importantly, pharmacological inhibition of MVI enhances the effect of hormone therapy, resulting in greater disruption of ER function than monotherapy alone. Moreover, MVI inhibition also suppresses the activity of hormone-resistant ER mutants, highlighting its potential to overcome therapy resistance. Our findings establish MVI as a critical regulator of ER nuclear dynamics and gene expression, supporting its candidacy as a novel therapeutic target in ER-positive breast cancer.

cancer biology↗

bioRxiv2025

In-cell structure of a LINC complex reveals the molecular basis for membrane remodelling and head-to-tail coupling in sperm cells

In eukaryotic cells, LINC complexes physically bridge the two nuclear membranes to cytoskeletal filaments, transmitting mechanical forces to the nucleus. Due to their dynamic and membrane-embedded nature, their architecture in native membranes has been hitherto elusive. Here, we combine in-situ electron cryo-tomography, AlphaFold predictions and molecular dynamics simulations to determine the sub-nanometer-resolution architecture of the trimeric [~]130 kDa SUN5-Nesprin3 LINC complex in human spermatozoa. The structure reveals a hexagonal lattice that stitches the nuclear envelope, creating a rigid interface that links the sperm nucleus to the flagellum. Our structure unveils a unique membrane-anchoring mechanism: a SUN5 KASH-lid {beta}-hairpin and a Nesprin3 amphipathic helix insert into the outer nuclear membrane, leading to cooperative remodelling of the nuclear envelope shape by the extended LINC lattice, which results in flattening the caudal sperm nucleus. Our integrative data provides key insights into the architecture and role of a LINC complex in situ, revealing molecular details of a severe male infertility phenotype.

biochemistry↗

bioRxiv2025

Nuclear Disruption as a Mechanism of the Chemotherapeutic Drug Paclitaxel in Interphase

Regulation of Lamin A/C levels and distribution is crucial for nuclear integrity and mechanotransduction via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Dysregulation of Lamin A/C correlates with poor cancer prognosis, and its levels determine sensitivity to the microtubule-stabilising drug paclitaxel. Paclitaxel is well-known for disrupting mitosis, yet it also reduces tumour size in slow-dividing tumours, indicating an additional, poorly characterised interphase mechanism. Here, we reveal that paclitaxel induces nuclear aberrations in interphase through SUN2-dependent Lamin A/C disruption. Using advanced optical imaging and electron cryo-tomography, we show the formation of aberrant microtubule-vimentin bundles during paclitaxel treatment, which coincides with nuclear deformation and altered Lamin A/C protein levels and organisation at the nuclear envelope. SUN2 is required for Lamin A/C reduction in paclitaxel and is in turn regulated by polyubiquitination. Furthermore, Lamin A/C expression levels determine not only cell survival during treatment but also recovery after drug removal. Our findings support a model in which paclitaxel acts through both defective mitosis and interphase nuclear-cytoskeletal disruption, providing additional mechanistic insights into a widely used anticancer drug.

cell biology↗

bioRxiv2024

Human spermatogenesis leads to a reduced nuclear pore structure and function

Nuclear pore complexes (NPCs) are nuclear gateways which regulate the transit of molecules larger than 40kDa through a Ran-dependent transport1. The somatic human NPC scaffold consists of three stacked rings: the cytoplasmic (CR), the nucleoplasmic (NR), and the inner ring (IR), which define a central channel approximately 55nm wide2. Although many studies have investigated human NPC architecture3, it remains largely unknown how the NPC accommodates the different functions of non-somatic cells. Here, we reveal the in-cell architecture of the human sperm NPC. We show that it exhibits a central channel less than 40nm-wide, outlined exclusively by the IR. This structural alteration is accompanied by a six-fold reduction in nuclear diffusion rate and mis-localization of Ran-dependent transport components. Additionally, we identify a network of septin filaments interconnecting NPCs within the inter-membrane space of the nuclear envelope (NE), suggesting a potential mechanical role in channel constriction. Furthermore, our human tissue imaging data indicate meiosis as a pivotal differentiation stage driving architectural changes to the NPC. Given the critical role of the IR for successful spermatogenesis4,5, our work offers important insight into this fundamental biological process. Our study also demonstrates the power of an integrative approach by combining electron-cryo- tomography, super-resolution-light-microscopy and biochemical analysis, to elucidate macromolecular structure-function relations in human physiological contexts.

cell biology↗

bioRxiv2024

A characterization of mitotic and centrosomal defects in a continuum model of Breast Cancer

Errors in mitosis can contribute to aneuploidy and CIN and play a pivotal role in cancer. So the identification of altered mitotic regulators can contribute to the understanding of the development and progression of breast cancer. In the present study we used an in vitro model of disease progression (the MCF10A series of BC continuum) and analyzed the errors of chromosome segregation that occur during the progression of the disease. Our findings indicated that the MCF10A series exhibited several abnormalities in chromosome segregation and its frequency increased with the disease progression. These errors included anaphase lagging chromosomes, micronuclei, nuclear buds, nucleoplasmic bridges, errors of chromosome alignment, and centrosome loss/amplification. Moreover, the presence of centrosome amplification disrupted the proper orientation of the mitotic spindle, resulting in the generation asymmetrical cell lines and aneuploidy in the MCF10A series. Hyper stable kinetochore-microtubule (kt-MT) attachment was also found in premalignant, preinvasive, and invasive cell lines, which can also explain the presence of errors of chromosome alignment. The human transcriptome array also determined possible negative regulators of ciliogenesis that can explain the mechanism of chromosome missegregation that lead to CIN found in the MCF10A series. Collectively, these findings highlight the importance of mitotic defects in the progression of breast cancer.

cell biology↗

bioRxiv2024

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

The Ras homolog (Rho) small GTPases, via their role in regulating the actin cytoskeleton, coordinate diverse cellular functions including cell morphology, adhesion and motility, as well as cell cycle progression, survival and apoptosis. The upstream Rho regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) that been implicated in cell migration, cell-cell junction formation and the mitotic checkpoint. In this study we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4 depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, we find that TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4 depleted cells delay in G1 as a consequence of elevated levels of the G1/S inhibitor p21waf1/cip1 and that TEM4 depleted cells that progress through to mitosis, do so with decreased transcription of CCNB1 and thus attenuated levels of cyclin B. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signalling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.

cell biology↗

bioRxiv2022

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

biophysics↗

bioRxiv2021

Follow-up of a hospital cohort during the first 3,530 suspected cases of COVID-19 in Sao Jose do Rio Preto, Sao Paulo, Brazil

IntroductionIn a global context, COVID-19 is the most significant health threat in the present days, evidenced by the fact that, in just over four months, SARS-CoV-2 has spread to 171 countries, reaching a Pandemic status. Most patients with COVID-19 have a mild course of the disease. However, approximately 20% develop severe illness with a high mortality rate which is associated with age, comorbidities, and immunosuppression. Epidemiological studies are used to reveal the extent of viral spread in homes, communities, and hospitals. Thus, preventive and control measures can be established by the authorities. ObjectiveIn this study, patients with suspect COVID-19 symptoms who search for hospital care at the city of Sao Jose do Rio Preto (Sao Paulo, Brazil) were monitored, in order to identify the first case of this new disease in the region. In the first two months (March and April), more than 3000 individuals looked for the public and private health system with suspected respiratory symptoms, but only 164 (8.4%) were COVID-19 confirmed. ResultsFrom those, males (56.1%) and patients of the age distribution of 16-59 (91.2%), with diarrhea (22.2%), runny nose (25%), altered taste (15.9%), and anosmia (11.6%) presented statistical significance, although none comorbidities were related with COVID-19 occurrence. The odds ratio analysis supports this finding. Days of onset of symptoms are positively associated with whit viral load, and the same happens with the occurrence of symptoms (dyspnea and low saturation).

molecular biology↗

bioRxiv2021

A targeted and tuneable DNA damage tool using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-Strand Breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR associated protein 9) endonuclease enzyme, is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

cell biology↗

bioRxiv2020

Binding partners regulate unfolding of myosin VI to activate the molecular motor

Myosin VI is the only minus-end actin motor and is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single molecule imaging to investigate the impact of binding partners upon myosin VI molecular organisation and dynamics.

biophysics↗

bioRxiv2020

High throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

Mechanobiology is focused on how the physical forces and the mechanical properties of proteins, cells and tissues contribute to physiology and disease. While the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. The broad applicability and versatility of our approach has been demonstrated through in vitro force-induced enzymatic activity and conformation changes, along with force-induced receptor activation and their downstream signalling pathways in live cells. Overall, our methodology allows for the first-time ensemble biochemical and cell-based assays to be performed under force, in high throughput format. This novel approach would substantially add to the mechano-biological toolbox and increase the availability of mechanobiology measurements.

biophysics↗

bioRxiv2020

Nuclear myosin VI regulates the spatial organization of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability which underpin the function these transcription factories remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI in the nanoscale organisation of RNAPII. We reveal that myosin VI acts as the molecular anchor that holds RNAPII into transcription factories. Perturbation of myosin VI leads to the disruption of RNAPII localisation, changes in chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of myosin VI in the spatial regulation of gene expression during the rapid response to changes in the cellular environment.

cell biology↗

bioRxiv2019

Binding partner regulation of Myosin VI: Loss of tumour-suppressor Dab2 leads to enhanced activity of nuclear myosin

Myosin VI is involved in a variety of cellular processes ranging from endocytosis to transcription. This multi-functional potential is achieved through alternative isoform splicing and through the interaction with a diverse network of binding partners. However, the interplay between the two modes of regulation remains unexplored. To this end, we have compared two different binding partners, Dab2 and CALCOCO2/NDP52, and their interaction with two myosin VI splice isoforms. We found that both isoforms adopt an auto-inhibited state and are subsequently activated by binding partner association. However, differential regulation is achieved through a high and a low affinity binding motifs within myosin VI, with one isoform having the high affinity site blocked. This allows competition between partners and links isoform splicing with binding partner selectivity. Dab2 competition hinders the activity of nuclear myosin VI by preventing DNA binding and transcription. Moreover, re-introduction of Dab2 in the Dab2-deficient MCF-7 cells leads to a decrease in myosin VI-dependent estrogen receptor gene expression. We propose that the frequent loss of Dab2 during the onset of cancer enables a higher level of nuclear myosin VI activity, thereby driving the activity of the estrogen receptor to promote tumourgenesis.

cell biology↗