RESUMO
Recent research has highlighted the growing significance of the mechanical properties of cells and tissues in the proper execution of physiological functions within an organism; alterations to these properties can potentially result in various diseases. These mechanical properties can be assessed using various techniques that vary in spatial and temporal resolutions as well as applications. Due to the wide range of mechanical behaviors exhibited by cells and tissues, a singular mapping technique may be insufficient in capturing their complexity and nuance. Consequently, by utilizing a combination of methods-multimodal mechanical mapping-researchers can achieve a more comprehensive characterization of mechanical properties, encompassing factors such as stiffness, modulus, viscoelasticity, and forces. Furthermore, different mapping techniques can provide complementary information and enable the exploration of spatial and temporal variations to enhance our understanding of cellular dynamics and tissue mechanics. By capitalizing on the unique strengths of each method while mitigating their respective limitations, a more precise and holistic understanding of cellular and tissue mechanics can be obtained. Here, we spotlight Brillouin microscopy (BM) as a noncontact, noninvasive, and label-free mechanical mapping modality to be coutilized alongside established mechanical probing methods. This review summarizes some of the most widely adopted individual mechanical mapping techniques and highlights several recent multimodal approaches demonstrating their utility. We envision that future studies aim to adopt multimodal techniques to drive advancements in the broader realm of mechanobiology.
Assuntos
Microscopia , Humanos , Animais , Fenômenos BiomecânicosRESUMO
Bladder mechanical properties are critical for organ function and tissue homeostasis. Therefore, alterations of tissue mechanics are linked to disease onset and progression. This study aims to characterize the tissue elasticity of the murine bladder wall considering its different anatomical components, both in healthy conditions and in actinic cystitis, a state characterized by tissue fibrosis. Here, we exploit Brillouin microscopy, an emerging technique in the mechanobiology field that allows mapping tissue mechanics at the microscale, in non-contact mode and free of labeling. We show that Brillouin imaging of bladder tissues is able to recognize the different anatomical components of the bladder wall, confirmed by histopathological analysis, showing different tissue mechanical properties of the physiological bladder, as well as a significant alteration in the presence of tissue fibrosis. Our results point out the potential use of Brillouin imaging on clinically relevant samples as a complementary technique to histopathological analysis, deciphering complex mechanical alteration of each tissue layer of an organ that strongly relies on mechanical properties to perform its function.
Assuntos
Cistite , Microscopia , Camundongos , Animais , Bexiga Urinária/diagnóstico por imagem , Elasticidade , Cistite/diagnóstico por imagem , FibroseRESUMO
In this study, transmission electron microscopy atomic force microscopy, and surface enhanced Raman spectroscopy are combined through a direct imaging approach, to gather structural and chemical information of complex molecular systems such as ion channels in their original plasma membrane. Customized microfabricated sample holder allows to characterize Nav channels embedded in the original plasma membrane extracted from neuronal cells that are derived from healthy human induced pluripotent stem cells. The identification of the channels is accomplished by using two different approaches, one of them widely used in cryo-EM (the particle analysis method) and the other based on a novel Zernike Polynomial expansion of the images bitmap. This approach allows to carry out a whole series of investigations, one complementary to the other, on the same sample, preserving its state as close as possible to the original membrane configuration.
Assuntos
Células-Tronco Pluripotentes Induzidas , Canais de Sódio Disparados por Voltagem , Membrana Celular/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Análise Espectral , Canais de Sódio Disparados por Voltagem/químicaRESUMO
The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.
Assuntos
Anormalidades Múltiplas/genética , Núcleo Celular/fisiologia , Cromatina/metabolismo , Face/anormalidades , Haploinsuficiência/genética , Doenças Hematológicas/genética , Histona-Lisina N-Metiltransferase/genética , Doenças Vestibulares/genética , Células 3T3 , Animais , Linhagem Celular , Linhagem da Célula/genética , Condrócitos/citologia , Condrogênese/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Camundongos , Osteócitos/citologia , Osteogênese/genética , Proteínas do Grupo Polycomb/genética , Estresse MecânicoRESUMO
Protein-protein interactions regulate almost all cellular functions and rely on a fine tune of surface amino acids properties involved on both molecular partners. The disruption of a molecular association can be caused even by a single residue mutation, often leading to a pathological modification of a biochemical pathway. Therefore the evaluation of the effects of amino acid substitutions on binding, and the ad hoc design of protein-protein interfaces, is one of the biggest challenges in computational biology. Here, we present a novel strategy for computational mutation and optimization of protein-protein interfaces. Modeling the interaction surface properties using the Zernike polynomials, we describe the shape and electrostatics of binding sites with an ordered set of descriptors, making possible the evaluation of complementarity between interacting surfaces. With a Monte Carlo approach, we obtain protein mutants with controlled molecular complementarities. Applying this strategy to the relevant case of the interaction between Ferritin and Transferrin Receptor, we obtain a set of Ferritin mutants with increased or decreased complementarity. The extensive molecular dynamics validation of the method results confirms its efficacy, showing that this strategy represents a very promising approach in designing correct molecular interfaces.
RESUMO
The maintenance of redox homeostasis in the brain is critical for the prevention of the development of neurodegenerative diseases. Drugs acting on brain redox balance can be promising for the treatment of neurodegeneration. For more than four decades, dimethyl fumarate (DMF) and other derivatives of fumaric acid ester compounds have been shown to mitigate a number of pathological mechanisms associated with psoriasis and relapsing forms of multiple sclerosis (MS). Recently, DMF has been shown to exert a neuroprotective effect on the central nervous system (CNS), possibly through the modulation of microglia detrimental actions, observed also in multiple brain injuries. In addition to the hypothesis that DMF is linked to the activation of NRF2 and NF-kB transcription factors, the neuroprotective action of DMF may be mediated by the activation of the glutathione (GSH) antioxidant pathway and the regulation of brain iron homeostasis. This review will focus on the role of DMF as an antioxidant modulator in microglia processes and on its mechanisms of action in the modulation of different pathways to attenuate neurodegenerative disease progression.
RESUMO
Dimethyl fumarate (DMF) is the only available approved drug for first line treatment of multiple sclerosis (MS), a lethal condition impairing central nervous system (CNS). To date, however, little is known of its mechanisms of action. Only recently, it has been suggested that DMF exerts neuroprotective effects acting as an immunomodulator and that it may alter the activation state of microglia cells, crucial in MS pathogenesis. However, DMF effects on microglia functions are still not well determined. Here, we examine the effects of DMF treatment on microglia functional activities, as phenotype, morphology, processes motility and rearrangement, migration, ATP response and iron uptake in mouse primary microglia culture and acute hippocampal slices. We found that DMF treatment reduces microglia motility, downregulating functional response to ATP, increases ferritin uptake and pushes microglia towards an anti-inflammatory phenotype, thus reducing its proinflammatory reactivity in response to tissue damage. These results highlight the effects of this compound on microglia functions and provide new insights on the mechanism of action of DMF in MS treatment.
Assuntos
Fumarato de Dimetilo , Preparações Farmacêuticas , Animais , Encéfalo , Fumarato de Dimetilo/farmacologia , Homeostase , Imunossupressores/farmacologia , Ferro , Camundongos , MicrogliaRESUMO
The transferrin receptor 1 (TfR1) is one of the key regulators of iron homeostasis for most higher organisms. It mediates cellular iron import through a constitutive clathrin-dependent endocytosis mechanism and by recruiting iron- regulator proteins as transferrin, Hereditary Hemochromatosis factor (HFE) and serum ferritin in response to cellular demand. The receptor is also opportunistically exploited by several viruses and the malaria parasite as a preferential door for cell invasion. In this review, we analyze the structural information available for TfR1 and all its functional complexes to figure out how structural signals in a single receptor can guide the recognition of multiple ligands and how the conservation of key residues in TfR1 might have a role in iron uptake and cell infection.
Assuntos
Ligantes , Receptores da Transferrina/química , Animais , Sítios de Ligação , Humanos , Ferro/química , Ferro/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores da Transferrina/metabolismoRESUMO
Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.
Assuntos
Antígenos CD/química , Apoferritinas/química , Proteínas de Protozoários/química , Receptores da Transferrina/química , Receptores Virais/química , Transferrina/química , Proteínas do Envelope Viral/química , Antígenos CD/genética , Antígenos CD/metabolismo , Apoferritinas/genética , Apoferritinas/metabolismo , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Proteína da Hemocromatose/química , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Humanos , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transferrina/genética , Transferrina/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation.
Assuntos
Apoferritinas/química , Apoferritinas/metabolismo , Elementos da Série dos Lantanídeos/química , Animais , Apoferritinas/genética , Sítios de Ligação , Linhagem Celular Tumoral , Escherichia coli , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Engenharia de ProteínasRESUMO
In humans the steroid 5alpha-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.
RESUMO
Human ferritins have been extensively studied to be used as nanocarriers for diverse applications and could represent a convenient alternative for targeted delivery of anticancer drugs and imaging agents. However, the most relevant limitation to their applications is the need for highly acidic experimental conditions during the initial steps of particle/cargo assembly, a process that could affect both drug stability and the complete reassembly of the ferritin cage. To overcome this issue the unique assembly of Archaeoglobus fulgidus ferritin was genetically engineered by changing a surface exposed loop of 12 amino acids connecting B and C helices to mimic the sequence of the analogous human H-chain ferritin loop. This new chimeric protein was shown to maintain the unique, cation linked, association-dissociation properties of Archaeoglobus fulgidus ferritin occurring at neutral pH values, while exhibiting the typical human H-homopolymer recognition by the transferrin receptor TfR1. The chimeric protein was confirmed to be actively and specifically internalized by HeLa cells, thus representing a unique nanotechnological tool for cell-targeted delivery of possible payloads for diagnostic or therapeutic purposes. Moreover, it was demonstrated that the 12 amino acids' loop is necessary and sufficient for binding to the transferrin receptor. The three-dimensional structure of the humanized Archaeoglobus ferritin has been obtained both as crystals by X-ray diffraction and in solution by cryo-EM.