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1.
Traffic ; 22(4): 123-136, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33347683

RESUMEN

Retromer core complex is an endosomal scaffold that plays a critical role in orchestrating protein trafficking within the endosomal system. Here we characterized the effect of the Parkinson's disease-linked Vps35 D620N in the endo-lysosomal system using Vps35 D620N rescue cell models. Vps35 D620N fully rescues the lysosomal and autophagy defects caused by retromer knock-out. Analogous to Vps35 knock out cells, the endosome-to-trans-Golgi network transport of cation-independent mannose 6-phosphate receptor (CI-M6PR) is impaired in Vps35 D620N rescue cells because of a reduced capacity to form endosome transport carriers. Cells expressing the Vps35 D620N variant have altered endosomal morphology, resulting in smaller, rounder structures with less tubule-like branches. At the molecular level retromer incorporating Vps35 D620N variant has a decreased binding to retromer associated proteins wiskott-aldrich syndrome protein and SCAR homologue (WASH) and SNX3 which are known to associate with retromer to form the endosome transport carriers. Hence, the partial defects on retrograde protein trafficking carriers in the presence of Vps35 D620N represents an altered cellular state able to cause Parkinson's disease.


Asunto(s)
Enfermedad de Parkinson , Endosomas/metabolismo , Humanos , Lisosomas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Transporte de Proteínas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
2.
J Neurosci Res ; 99(6): 1515-1532, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33682204

RESUMEN

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co-localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction-limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super-resolution microscopy to the analysis of routine formalin-fixed paraffin sections of archival human brain. The investigation of microglia-synapse interactions in dementia will be of special interest in this context.


Asunto(s)
Microglía/fisiología , Microglía/ultraestructura , Microscopía/métodos , Sinapsis/fisiología , Sinapsis/ultraestructura , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Animales , Anticuerpos , Femenino , Humanos , Larva , Masculino , Ratones , Microscopía Confocal , Persona de Mediana Edad , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Fijación del Tejido , Pez Cebra
3.
Plant Cell Physiol ; 61(10): 1775-1787, 2020 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-32761075

RESUMEN

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.


Asunto(s)
Arabidopsis/ultraestructura , Pared Celular/ultraestructura , Colorantes Fluorescentes , Imagen Óptica/métodos , Arabidopsis/crecimiento & desarrollo , Membrana Celular/ultraestructura , Celulosa/metabolismo , Microscopía Confocal , Propidio , Rodamina 123
4.
Mol Cell Proteomics ; 15(8): 2537-53, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27174698

RESUMEN

The mitotic spindle is required for chromosome congression and subsequent equal segregation of sister chromatids. These processes involve a complex network of signaling molecules located at the spindle. The endocytic protein, clathrin, has a "moonlighting" role during mitosis, whereby it stabilizes the mitotic spindle. The signaling pathways that clathrin participates in to achieve mitotic spindle stability are unknown. Here, we assessed the mitotic spindle proteome and phosphoproteome in clathrin-depleted cells using quantitative MS/MS (data are available via ProteomeXchange with identifier PXD001603). We report a spindle proteome that consists of 3046 proteins and a spindle phosphoproteome consisting of 5157 phosphosites in 1641 phosphoproteins. Of these, 2908 (95.4%) proteins and 1636 (99.7%) phosphoproteins are known or predicted spindle-associated proteins. Clathrin-depletion from spindles resulted in dysregulation of 121 proteins and perturbed signaling to 47 phosphosites. The majority of these proteins increased in mitotic spindle abundance and six of these were validated by immunofluorescence microscopy. Functional pathway analysis confirmed the reported role of clathrin in mitotic spindle stabilization for chromosome alignment and highlighted possible new mechanisms of clathrin action. The data also revealed a novel second mitotic role for clathrin in bipolar spindle formation.


Asunto(s)
Clatrina/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Huso Acromático/metabolismo , Cromatografía Liquida , Células HeLa , Humanos , Metafase , Fosforilación , Unión Proteica , Mapas de Interacción de Proteínas , Transducción de Señal , Espectrometría de Masas en Tándem
5.
BMC Bioinformatics ; 18(Suppl 16): 566, 2017 12 28.
Artículo en Inglés | MEDLINE | ID: mdl-29297284

RESUMEN

BACKGROUND: Cell division (mitosis) results in the equal segregation of chromosomes between two daughter cells. The mitotic spindle plays a pivotal role in chromosome alignment and segregation during metaphase and anaphase. Structural or functional errors of this spindle can cause aneuploidy, a hallmark of many cancers. To investigate if a given protein associates with the mitotic spindle and regulates its assembly, stability, or function, fluorescence microscopy can be performed to determine if disruption of that protein induces phenotypes indicative of spindle dysfunction. Importantly, functional disruption of proteins with specific roles during mitosis can lead to cancer cell death by inducing mitotic insult. However, there is a lack of automated computational tools to detect and quantify the effects of such disruption on spindle integrity. RESULTS: We developed the image analysis software tool MatQuantify, which detects both large-scale and subtle structural changes in the spindle or DNA and can be used to statistically compare the effects of different treatments. MatQuantify can quantify various physical properties extracted from fluorescence microscopy images, such as area, lengths of various components, perimeter, eccentricity, fractal dimension, satellite objects and orientation. It can also measure textual properties including entropy, intensities and the standard deviation of intensities. Using MatQuantify, we studied the effect of knocking down the protein clathrin heavy chain (CHC) on the mitotic spindle. We analysed 217 microscopy images of untreated metaphase cells, 172 images of metaphase cells transfected with small interfering RNAs targeting the luciferase gene (as a negative control), and 230 images of metaphase cells depleted of CHC. Using the quantified data, we trained 23 supervised machine learning classification algorithms. The Support Vector Machine learning algorithm was the most accurate method (accuracy: 85.1%; area under the curve: 0.92) for classifying a spindle image. The Kruskal-Wallis and Tukey-Kramer tests demonstrated that solidity, compactness, eccentricity, extent, mean intensity and number of satellite objects (multipolar spindles) significantly differed between CHC-depleted cells and untreated/luciferase-knockdown cells. CONCLUSION: MatQuantify enables automated quantitative analysis of images of mitotic spindles. Using this tool, researchers can unambiguously test if disruption of a protein-of-interest changes metaphase spindle maintenance and thereby affects mitosis.


Asunto(s)
Mitosis/genética , Huso Acromático/clasificación , Humanos
6.
J Cell Sci ; 128(4): 755-67, 2015 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-25588841

RESUMEN

Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/fisiología , Receptores ErbB/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Nexinas de Clasificación/metabolismo , Vesículas Transportadoras/metabolismo , Ubiquitinación , Proteínas de Transporte Vesicular/genética
7.
Microbiol Spectr ; 10(5): e0241922, 2022 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-36102643

RESUMEN

Cell wall deficient bacterial L-forms are induced by exposure to cell wall-targeting antibiotics and immune effectors such as lysozyme. L-forms of different bacteria (including Escherichia coli) have been reported in human infections, but whether this is a normal adaptive strategy or simply an artifact of antibiotic treatment in certain bacterial species remains unclear. Here we show that members of a representative, diverse set of pathogenic E. coli readily proliferate as L-forms in supratherapeutic concentrations of the broad-spectrum antibiotic meropenem. We report that they are completely resistant to antibiotics targeting any penicillin-binding proteins in this state, including PBP1A/1B, PBP2, PBP3, PBP4, and PBP5/6. Importantly, we observed that reversion to the cell-walled state occurs efficiently, less than 20 h after antibiotic cessation, with few or no changes in DNA sequence. We defined for the first time a logarithmic L-form growth phase with a doubling time of 80 to 190 min, followed by a stationary phase in late cultures. We further demonstrated that L-forms are metabolically active and remain normally susceptible to antibiotics that affect DNA torsion and ribosomal function. Our findings provide insights into the biology of L-forms and help us understand the risk of ß-lactam failure in persistent infections in which L-forms may be common. IMPORTANCE Bacterial L-forms require specialized culture techniques and are neither widely reported nor well understood in human infections. To date, most of the studies have been conducted on Gram-positive and stable L-form bacteria, which usually require mutagenesis or long-term passages for their generation. Here, using an adapted osmoprotective growth media, we provide evidence that pathogenic E. coli can efficiently switch to L-forms and back to a cell-walled state, proliferating aerobically in supratherapeutic concentrations of antibiotics targeting cell walls with few or no changes in their DNA sequences. Our work demonstrates that L-form switching is an effective adaptive strategy in stressful environments and can be expected to limit the efficacy of ß-lactam for many important infections.


Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacología , Resistencia betalactámica , beta-Lactamas/farmacología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Meropenem/farmacología , Muramidasa/farmacología , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Proteínas de Unión a las Penicilinas/farmacología
8.
Fluids Barriers CNS ; 19(1): 14, 2022 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-35135574

RESUMEN

BACKGROUND: Cerebrospinal fluid (CSF) circulation in the brain has garnered considerable attention in recent times. In contrast, there have been fewer studies focused on the spine, despite the expected importance of CSF circulation in disorders specific to the spine, including syringomyelia. The driving forces that regulate spinal CSF flow are not well defined and are likely to be different to the brain given the anatomical differences and proximity to the heart and lungs. The aims of this study were to determine the effects of heart rate, blood pressure and respiration on the distribution of CSF tracers in the spinal subarachnoid space, as well as into the spinal cord interstitium. METHODS: In Sprague Dawley rats, physiological parameters were manipulated such that the effects of spontaneous breathing (generating alternating positive and negative intrathoracic pressures), mechanical ventilation (positive intrathoracic pressure only), tachy/bradycardia, as well as hyper/hypotension were separately studied. To investigate spinal CSF hydrodynamics, in vivo near-infrared imaging of intracisternally infused indocyanine green was performed. CSF tracer transport was further characterised with in vivo two-photon intravital imaging. Tracer influx at a microscopic level was quantitatively characterised by ex vivo epifluorescence imaging of fluorescent ovalbumin. RESULTS: Compared to mechanically ventilated controls, spontaneous breathing animals had significantly greater movement of tracer in the subarachnoid space. There was also greater influx into the spinal cord interstitium. Hypertension and tachycardia had no significant effect on spinal subarachnoid spinal CSF tracer flux and exerted less effect than respiration on tracer influx into the spinal cord. CONCLUSIONS: Intrathoracic pressure changes that occur over the respiratory cycle, particularly decreased intrathoracic pressures generated during inspiration, have a profound effect on tracer movement after injection into spinal CSF and increase cord parenchymal tracer influx. Arterial pulsations likely drive fluid transport from perivascular spaces into the surrounding interstitium, but their overall impact is less than that of the respiratory cycle on net tracer influx.


Asunto(s)
Presión Sanguínea/fisiología , Líquido Cefalorraquídeo/fisiología , Frecuencia Cardíaca/fisiología , Respiración , Médula Espinal/fisiología , Tórax/fisiología , Animales , Hidrodinámica , Masculino , Ratas Sprague-Dawley , Respiración Artificial , Coloración y Etiquetado , Espacio Subaracnoideo/fisiología
9.
Phys Chem Chem Phys ; 12(15): 3753-61, 2010 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-20358070

RESUMEN

The first passage probability (FPP), of trafficked intracellular particles reaching a displacement L, in a given time t or inverse velocity S = t/L, can be calculated robustly from measured particle tracks. The FPP gives a measure of particle movement in which different types of motion, e.g. diffusion, ballistic motion, and transient run-rest motion, can readily be distinguished in a single graph, and compared with mathematical models. The FPP is attractive in that it offers a means of reducing the data in the measured tracks, without making assumptions about the mechanism of motion. For example, it does not employ smoothing, segmentation or arbitrary thresholds to discriminate between different types of motion in a particle track. In contrast to conventional mean square displacement analysis, FPP is sensitive to a small population of trafficked particles that move long distances (> or = 5 microm), which are thought to be crucial for efficient long range signaling in theories of network dynamics. Taking experimental data from tracked endocytic vesicles, and calculating the FPP, we see how molecular treatments affect the trafficking. We show the FPP can quantify complicated movement which is neither completely random nor completely deterministic, making it highly applicable to trafficked particles in cell biology.


Asunto(s)
Modelos Teóricos , Algoritmos , Difusión , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Probabilidad , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
10.
Oncogenesis ; 8(5): 29, 2019 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-30988276

RESUMEN

Micronuclei represent the cellular attempt to compartmentalize DNA to maintain genomic integrity threatened by mitotic errors and genotoxic events. Some micronuclei show aberrant nuclear envelopes (NEs) that collapse, generating damaged DNA that can promote complex genome alterations. However, ruptured micronuclei also provide a pool of cytosolic DNA that can stimulate antitumor immunity, revealing the complexity of micronuclear impact on tumor progression. The ESCRT-III (Endosomal Sorting Complex Required for Transport-III) complex ensures NE reseals during late mitosis and is repaired in interphase. Therefore, ESCRT-III activity maybe crucial for maintaining the integrity of other genomic structures enclosed by a NE. ESCRT-III activity at the NE is coordinated by the subunit CHMP7. We show that CHMP7 and ESCRT-III protect against the genomic instability associated with micronuclei formation. Loss of ESCRT-III activity increases the population of micronuclei with ruptured NEs, revealing that its NE repair activity is also necessary to maintain micronuclei integrity. Surprisingly, aberrant accumulation of ESCRT-III are found at the envelope of most acentric collapsed micronuclei, suggesting that ESCRT-III is not recycled efficiently from these structures. Moreover, CHMP7 depletion relieves micronuclei from the aberrant accumulations of ESCRT-III. CHMP7-depleted cells display a reduction in micronuclei containing the DNA damage marker RPA and a sensor of cytosolic DNA. Thus, ESCRT-III activity appears to protect from the consequence of genomic instability in a dichotomous fashion: ESCRT-III membrane repair activity prevents the occurrence of micronuclei with weak envelopes, but the aberrant accumulation of ESCRT-III on a subset of micronuclei appears to exacerbate DNA damage and sustain proinflammatory pathways.

11.
PLoS One ; 6(9): e24479, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21915335

RESUMEN

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85-98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150(Glued) to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150(Glued) remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms(-1) that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.


Asunto(s)
Dineínas/metabolismo , Endosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Complejo Dinactina , Dineínas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Reacción en Cadena de la Polimerasa , Interferencia de ARN , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo
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