RESUMO
Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Fator de Processamento U2AF , Grânulos de Estresse , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/genética , Sítios de Splice de RNA , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Grânulos de Estresse/metabolismoRESUMO
Understanding cellular organization demands the best possible spatial resolution in all three dimensions. In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved iso-stimulated emission depletion nanoscope, which uses an advanced adaptive optics strategy to achieve sub-50-nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.
Assuntos
Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Óptica e Fotônica/métodos , Imageamento Tridimensional , Razão Sinal-RuídoRESUMO
Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.
Assuntos
Imagem Óptica , Frações Subcelulares/metabolismo , Animais , Humanos , Organelas/metabolismoRESUMO
Activation-induced cytidine deaminase (AID) initiates DNA double-strand breaks (DSBs) in the IgH gene (Igh) to stimulate isotype class switch recombination (CSR), and widespread breaks in non-Igh (off-target) loci throughout the genome. Because the DSBs that initiate class switching occur during the G1 phase of the cell cycle, and are repaired via end joining, CSR is considered a predominantly G1 reaction. By contrast, AID-induced non-Igh DSBs are repaired by homologous recombination. Although little is known about the connection between the cell cycle and either induction or resolution of AID-mediated non-Igh DSBs, their repair by homologous recombination implicates post-G1 phases. Coordination of DNA breakage and repair during the cell cycle is critical to promote normal class switching and prevent genomic instability. To understand how AID-mediated events are regulated through the cell cycle, we have investigated G1-to-S control in AID-dependent genome-wide DSBs. We find that AID-mediated off-target DSBs, like those induced in the Igh locus, are generated during G1. These data suggest that AID-mediated DSBs can evade G1/S checkpoint activation and persist beyond G1, becoming resolved during S phase. Interestingly, DSB resolution during S phase can promote not only non-Igh break repair, but also Ig CSR. Our results reveal novel cell cycle dynamics in response to AID-initiated DSBs, and suggest that the regulation of the repair of these DSBs through the cell cycle may ensure proper class switching while preventing AID-induced genomic instability.
Assuntos
Citidina Desaminase/fisiologia , Quebras de DNA de Cadeia Dupla , Switching de Imunoglobulina/genética , Isotipos de Imunoglobulinas/genética , Fase S/genética , Fase S/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Reparo do DNA/genética , Reparo do DNA/imunologia , Fase G1/genética , Fase G1/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.
Assuntos
Biofísica/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Biofísica/economia , Biofísica/instrumentação , Fluoresceína/farmacologia , Corantes Fluorescentes/farmacologia , Aumento da Imagem , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/instrumentação , Lasers , Luz , Microscopia/métodos , Microscopia de Fluorescência/instrumentação , SoftwareRESUMO
The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and analyze the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a troubleshooting guide and present examples of anticipated results. An experienced optical instrument builder will require ~12 months from the start of ordering hardware components to acquiring high-quality biological images.
Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , HumanosRESUMO
The major histocompatibility complex (MHC) of the type 1 diabetes-prone NOD mouse lacks a functional class II H2-Ea gene such that antigen presenting cells (APCs) are I-E null. Transgenic expression of Ea in NOD mice both restores I-E expression and confers complete protection from diabetes progression. Non-myeloablative neonatal transplantation of bone marrow cells from such I-E+ transgenic donors into NOD recipients resulted in low-level but long-term haematopoietic stem cell (HSC) engraftment. Despite low levels of I-E antigen expression in blood (averaging 0.4-3.8% of total MHC class II-positive population), chimeric recipients were protected from overt diabetes, although not insulitis development. Adoptive transfer of diabetes into immunodeficient NOD-Rag recipients that received chimeric splenocytes from primary recipients confirmed the presence of an autoreactive T cell repertoire. The demonstration that purified T cells from these weak chimeras were not tolerant to irradiated transgenic I-E+ splenocytes indicated that I-E+ donor cells provide a constant, low-level immune stimulation capable of up-regulating nominally deficient immunoregulatory networks. This study raises the possibility that cord blood HSCs from infants with high risk HLA haplotypes and a family history of type 1 diabetes might be re-introduced without myoablative treatments following transfection with a single HLA class II allele associated with diabetes resistance.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Alelos , Animais , Transplante de Medula Óssea , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fatores SexuaisRESUMO
The majority of swollen polymer networks exhibit spatial variations in crosslink density. These spatial heterogeneities are particularly important in colloidal gel particles, or microgels, where they manifest themselves on the nanoscale and impact mechanical and transport properties. Despite their importance, the real space nanostructure of these heterogeneities at the individual particle level has remained elusive. Using state of the art super-resolution microscopy known as Whole cell 4Pi Single Molecule Switching Nanoscopy (W-4PiSMSN) we demonstrate 3D nanoscale mapping of spatial crosslink heterogeneities in a model system of poly(N-isopropylacrylamide) colloidal gel particles containing a novel fluorophore tagged crosslinker. We reveal the presence of higher crosslink density clusters embedded in a lower crosslink density matrix within the core of individual microgel particles, a phenomenon that has been predicted, but never been observed before in real space. The morphology of the clusters provides insight into the kinetics of microgel formation. This study also provides proof-of-concept 3D super-resolution imaging of spatial heterogeneities in bulk hydrogels.
RESUMO
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (â¼3 µm), thin (â¼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.
Assuntos
Fator 1 de Ribosilação do ADP/fisiologia , Complexo de Golgi/fisiologia , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Clatrina/metabolismo , Complexo I de Proteína do Envoltório/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Membranas Intracelulares/metabolismoRESUMO
OBJECTIVE: The goal of this study was to determine if competitive pressure was placed on hematopoietic stem cells (HSC) by a coinjected "carrier" population that maintains short-term survival of the host. Our hypothesis was that delayed introduction of "carrier" cells would increase engraftment of donor HSC. MATERIALS AND METHODS: Competitive repopulation assays were performed using genetically distinguishable whole bone marrow (BM) populations. Donor BM was competed against carrier BM that was coinjected or injected 3 or 4 days later. Radioprotection with delayed carrier injection also was examined by performing the initial HSC transplantation with Hoechst(lo) side population (SP) cells. SP HSC incubated with cytokines and BM stroma to stimulate cell cycling before transplantation also were tested using coinjection or delayed carrier administration. RESULTS: Delayed introduction of carrier whole BM increased peripheral expansion of donor whole BM, freshly isolated HSC, or cytokine-stimulated HSC compared to coinjection with carrier cells. A 3-day delay in carrier administration maintained radioprotection in 100% of lethally irradiated recipients of highly enriched HSC, whereas a 4-day delay did not rescue these recipients from death. When recipients are rescued, recovering host marrow can compete against donor HSC unless sufficient donor cells are injected. CONCLUSIONS: Delayed introduction of carrier BM significantly increases donor HSC engraftment and peripheral expansion by reducing competition in the host. Competition by a coinjected carrier cell population or recovery of host marrow significantly reduces the therapeutic efficacy of normal or in vitro manipulated donor HSC.
Assuntos
Transplante de Medula Óssea/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante Homólogo/métodos , Animais , Benzimidazóis/análise , Divisão Celular , Linhagem da Célula , Modelos Animais de Doenças , Corantes Fluorescentes/análise , Glucuronidase/deficiência , Glucuronidase/genética , Glucuronidase/fisiologia , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/classificação , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Mutantes , Mucopolissacaridose VII/genética , Mucopolissacaridose VII/terapia , Tolerância a Radiação , Fatores de TempoRESUMO
We present, for the first time to our knowledge, quantitative phase images associated with unstained 5 mum thick tissue slices of mouse brain, spleen, and liver. The refractive properties of the tissue are retrieved in terms of the average refractive index and its spatial variation. We find that the average refractive index varies significantly with tissue type, such that the brain is characterized by the lowest value and the liver by the highest. The spatial power spectra of the phase images reveal power law behavior with different exponents for each tissue type. This approach opens a new possibility for stain-free characterization of tissues, where the diagnostic power is provided by the intrinsic refractive properties of the biological structure. We present results obtained for liver tissue affected by a lysosomal storage disease and show that our technique can quantify structural changes during this disease development.
Assuntos
Encéfalo/patologia , Interpretação de Imagem Assistida por Computador/métodos , Fígado/patologia , Microscopia de Contraste de Fase/instrumentação , Refratometria/instrumentação , Baço/patologia , Animais , Mapeamento Encefálico , Modelos Animais de Doenças , Hepatopatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase/métodos , Modelos Estatísticos , Refratometria/métodos , Distribuição TecidualRESUMO
Treatment of nonmalignant childhood disorders by bone marrow transplantation (BMT) is limited by toxicity from preparatory regimens and immune consequences associated with engraftment of allogeneic donor cells. Using costimulatory blockade (anti-CD40L mAb and CTLA-4Ig) combined with high-dose BMT in nonablated neonates, we obtained engraftment and established tolerance using both partially MHC mismatched (H2g7 into H2b) and fully mismatched BM (H2s into H2b). Recipients were mucopolysaccharidosis type VII (MPS VII) mice with lysosomal storage disease in order to assess therapeutic outcome. Recipients treated with donor lymphocyte infusion (DLI) amplified microchimerism to full donor. Recipients without DLI maintained long-term engraftment, tolerance, and had extended life spans. DLI increased donor cell mediated replacement of beta-glucuronidase (GUSB) activity in all tissues and maintained clearance of lysosomes better than in non-DLI-treated mice. DLI amplification of partially mismatched BM and fully mismatched BM caused late onset chronic GvHD in 56% and 100% of recipients, respectively.
Assuntos
Transplante de Medula Óssea , Ativação Linfocitária , Transfusão de Linfócitos , Doenças por Armazenamento dos Lisossomos/terapia , Agonistas Mieloablativos , Animais , Quimera , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Cardiopatias/terapia , Imuno-Histoquímica , Rim/patologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Transplante HomólogoRESUMO
A significant number of nonmalignant, progressive childhood disorders respond to bone marrow transplantation (BMT). Toxic myeloablative pretreatment regimens, graft failure, and graft-vs-host disease complicate the utility of BMT for neonatal treatment. We recently demonstrated high-dose BMT in neonatal animals enables chimeric engraftment without toxic myeloablation. Reagents that block T cell costimulation (anti-CD40L mAb and/or CTLA-4Ig) establish tolerant allogeneic engraftment in adult recipients. Donor lymphocyte infusion (DLI) re-establishes failing grafts and treats malignant relapse via a graft-vs-leukemia response. In this study, we tested the hypothesis that combining these approaches would allow tolerant allogeneic engraftment devoid of myeloablation in neonatal normal and mutant mice with lysosomal storage disease. Tolerant chimeric allogeneic engraftment was achieved before DLI only in the presence of both anti-CD40L mAb and CTLA-4Ig. DLI amplified allografts to full donor engraftment long-term. DLI-treated mice either maintained long-term tolerance or developed late-onset chronic graft-vs-host disease. This combinatorial approach provides a nontoxic method to establish tolerant allogeneic engraftment for treatment of progressive childhood diseases.
Assuntos
Animais Recém-Nascidos/imunologia , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Transplante de Medula Óssea/imunologia , Ligante de CD40/imunologia , Imunoconjugados/administração & dosagem , Ativação Linfocitária/imunologia , Condicionamento Pré-Transplante/métodos , Abatacepte , Animais , Animais Recém-Nascidos/genética , Transplante de Medula Óssea/métodos , Transplante de Medula Óssea/patologia , Ligante de CD40/fisiologia , Células Cultivadas , Quimera/imunologia , Doença Crônica , Quimioterapia Combinada , Feminino , Facilitação Imunológica de Enxerto/métodos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Antígenos H-2/genética , Humanos , Tolerância Imunológica/genética , Ativação Linfocitária/genética , Transfusão de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Mutantes , Transplante HomólogoRESUMO
Lysosomal storage diseases (LSD) respond to bone marrow (BM) transplantation when donor-derived cells deliver needed enzyme. Hypothetically, the ubiquitous resident macrophages (MPhi) are the primary delivery vehicle of therapeutic protein. In mucopolysaccharidosis type VII (MPS VII) mice with LSD, transplanted mature MPhi reduce undegraded glycosaminoglycans (GAG) in the lysosome but are incapable of self-renewal, leading to return of storage after 1 month. We show here that a population of early BM-derived myeloid progenitors devoid of long-term hematopoietic stem cells (LT-HSC) engrafted MPS VII BM, released monocytes into peripheral blood (PBL), and engrafted tissues at known sites of resident MPhi. These primitive Mac-1- cells were sorted from normal whole BM and were defined by ER-MP12hi20-58med/hi labeling. Lysosomal storage was reduced in liver, spleen, thymus, heart, kidney, and bone. Cells persisted for 3 months, suggesting self-renewal capacity or a long half-life. Cells sorted from BM by ER-MP12-20hi marker expression (which are maturer myeloid cells that express Mac-1) engrafted tissues instead of BM and quantitatively repopulated less than cells derived from the ER-MP12hi20-58med/hi population. Also, reduction of lysosomal storage was variable and generally less when compared to that following transplantation of immature ER-MP12hi20-58med/hi cells. We conclude that primitive myeloid progenitors are more therapeutic for LSD than mature myeloid cells due to their greater longevity and increased capacity to seed tissues. The ability of cells derived from these primitive precursors to seed deep within tissues make them excellent candidates for both cellular therapy and gene transfer techniques to cure a wide range of metabolic diseases.