RESUMEN
Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet microscopy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of "harmonic balanced" lattice light sheet that improves performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.
RESUMEN
Professor Bruce Ames demonstrated that nutritional recommendations should be adjusted in order to 'tune-up' metabolism and reduce mitochondria decay, a hallmark of aging and many disease processes. A major subset of tunable nutrients are the minerals, which despite being integral to every aspect of metabolism are often deficient in the typical Western diet. Mitochondria are particularly rich in minerals, where they function as essential cofactors for mitochondrial physiology and overall cellular health. Yet substantial knowledge gaps remain in our understanding of the form and function of these minerals needed for metabolic harmony. Some of the minerals have known activities in the mitochondria but with incomplete regulatory detail, whereas other minerals have no established mitochondrial function at all. A comprehensive metallome of the mitochondria is needed to fully understand the patterns and relationships of minerals within metabolic processes and cellular development. This brief overview serves to highlight the current progress towards understanding mineral homeostasis in the mitochondria and to encourage more research activity in key areas. Future work may likely reveal that adjusting the amounts of specific nutritional minerals has longevity benefits for human health.
Asunto(s)
Longevidad , Mitocondrias , Diferenciación Celular , Humanos , Minerales/metabolismo , Mitocondrias/metabolismo , Oxidación-ReducciónRESUMEN
This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies. Compared with MHz impedance cytometers, operating at GHz frequencies offers direct intracellular permittivity probing due to electric fields penetrating through the cellular membrane. To overcome the detection challenges at high frequencies, the spectrometer employs on-chip oscillator-based sensors, which embeds simultaneous frequency generation, electrode excitation, and signal detection capabilities. By employing an injection-locking phase-detection technique, the spectrometer offers state-of-the-art sensitivity, achieving a less than 1 aFrms capacitance detection limit (or 5 ppm in frequency-shift) at a 100 kHz noise filtering bandwidth, enabling high throughput (>1k cells per s), with a measured cellular SNR of more than 28 dB. With CMOS/microfluidics co-design, we distribute four sensing channels at 6.5, 11, 17.5, and 30 GHz in an arrayed format whereas the frequencies are selected to center around the water relaxation frequency at 18 GHz. An issue in the integration of CMOS and microfluidics due to size mismatch is also addressed through introducing a cost-efficient epoxy-molding technique. With 3-D hydrodynamic focusing microfluidics, we perform characterization on four different cell lines including two breast cell lines (MCF-10A and MDA-MB-231) and two leukocyte cell lines (K-562 and THP-1). After normalizing the higher frequency signals to the 6.5 GHz ones, the size-independent dielectric opacity shows a differentiable distribution at 17.5 GHz between normal (0.905 ± 0.160, mean ± std.) and highly metastatic (1.033 ± 0.107) breast cells with p ⪠0.001.
Asunto(s)
Espectroscopía Dieléctrica/instrumentación , Citometría de Flujo/instrumentación , Dispositivos Laboratorio en un Chip , Metales/química , Microondas , Semiconductores , Análisis de la Célula Individual/instrumentación , Línea Celular Tumoral , Humanos , Óxidos/químicaRESUMEN
Microtentacles are thin, flexible cell protrusions that have recently been described and whose presence enhances efficient attachment of circulating cells. They are found on circulating tumor cells and can be induced on a wide range of breast cancer cell lines, where they are promoted by factors that either stabilize microtubules or destabilize the actin cytoskeleton. Evidence suggests that they are relevant to the metastatic spread of cancer, so understanding their structure and formation may lead to useful therapies. Microtentacles are formed by microtubules and contain vimentin intermediate filaments, but beyond this, there is little information about their ultrastructure. We have used electron microscopy of high pressure frozen sections and tomography of cryo-prepared intact cells, along with super resolution fluorescence microscopy, to provide the first ultrastructural insights into microtubule and intermediate filament arrangement within microtentacles. By scanning electron microscopy it was seen that microtentacles form within minutes of addition of drugs that stabilize microtubules and destabilize actin filaments. Mature microtentacles were found to be well below one micrometer in diameter, tapering gradually to below 100nm at the distal ends. They also contained frequent branches and bulges suggestive of heterogeneous internal structure. Super resolution fluorescence microscopy and examination of sectioned samples showed that the microtubules and intermediate filaments can occupy different areas within the microtentacles, rather than interacting intimately as had been expected. Cryo-electron tomography of thin regions of microtentacles revealed densely packed microtubules and absence of intermediate filaments. The number of microtubules ranged from several dozen in some areas to just a few in the thinnest regions, with none of the regular arrangement found in axonemes. Improved understanding of the mechanism of microtentacle formation, as well as the resultant structure, will be valuable in developing therapies against metastasis, if the hypothesized role of microtentacles in metastasis is confirmed. This work provides a significant step in this direction.
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Fenómenos Fisiológicos Celulares/fisiología , Filamentos Intermedios/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Animales , Línea Celular Tumoral , Humanos , Filamentos Intermedios/ultraestructura , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Vimentina/metabolismoRESUMEN
Poor consistency of the ice thickness from one area of a cryo-electron microscope (cryo-EM) specimen grid to another, from one grid to the next, and from one type of specimen to another, motivates a reconsideration of how to best prepare suitably thin specimens. Here we first review the three related topics of wetting, thinning, and stability against dewetting of aqueous films spread over a hydrophilic substrate. We then suggest that the importance of there being a surfactant monolayer at the air-water interface of thin, cryo-EM specimens has been largely underappreciated. In fact, a surfactant layer (of uncontrolled composition and surface pressure) can hardly be avoided during standard cryo-EM specimen preparation. We thus suggest that better control over the composition and properties of the surfactant layer may result in more reliable production of cryo-EM specimens with the desired thickness.
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Microscopía por Crioelectrón , Tensoactivos/química , Aire , Estabilidad de Medicamentos , Electrones , Presión , Solventes/química , Volatilización , Agua/química , HumectabilidadRESUMEN
Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G(1)/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22(ts), encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22(ts) we found that stabilized microtubules or increased bundling can rescue cut7-22(ts) associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on gamma-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to gamma-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/gamma-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22(ts) strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.
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Polaridad Celular/fisiología , Centro Organizador de los Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Sitios de Unión/fisiología , Secuencia Conservada/genética , Cinesinas/genética , Cinesinas/metabolismo , Sustancias Macromoleculares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/ultraestructura , Mutación/genética , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Schizosaccharomyces/genética , Schizosaccharomyces/ultraestructura , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/ultraestructura , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Chemotherapeutics used to treat prostate cancer are often from a class of drugs that target microtubule networks, such as paclitaxel. A previous report indicated that supplemental zinc sensitized prostate cancer cells to paclitaxel-induced apoptosis, suggesting that increased zinc levels might enhance paclitaxel efficacy. The effect of zinc deficiency on paclitaxel activity is not known though, so we tested this in two prostate cancer cell lines maintained under moderately zinc-deficient conditions. LNCaP and PC3 cell lines were used as models of early and late-stage prostate cancer, respectively. Cells cultured in reduced zinc levels did not demonstrate altered cell viability, growth rates, or intracellular zinc content. Additionally, zinc deficiency alone had no apparent effect on cell cycle kinetics or apoptosis levels. However, the IC(50) for paclitaxel-induced cell cycle arrest increased in LNCaP cells from zinc-deficient compared to zinc-replete conditions. Consequently, paclitaxel-induced apoptosis was reduced in LNCaP cells from zinc-deficient compared to zinc-replete conditions. In PC3 cells, the effects of paclitaxel were independent of zinc status. Reduced extracellular zinc levels were shown to affect paclitaxel activity in a prostate cancer cell line. Given the prevalence of zinc deficiency, determining how chemotherapeutic action is modulated by zinc adequacy may have clinical importance.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Paclitaxel/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Zinc/deficiencia , Ciclo Celular/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Masculino , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The microtubule cytoskeleton is involved in regulation of cell morphology, differentiation, and cell cycle progression. Precisely controlled dynamic properties are required for these microtubule functions. To better understand how tubulin's dynamics are embedded in its primary sequence, we investigated in vivo the consequences of altering a single, highly conserved residue in beta-tubulin that lies at the interface between two structural domains. The residue differs between the cold-adapted Antarctic fish and temperate animals in a manner that suggests a role in microtubule stability. Fungi, like the Antarctic fish, have a phenylalanine in this position, whereas essentially all other animals have tyrosine. We mutated the corresponding residue in fission yeast to tyrosine. Temperature effects were subtle, but time-lapse microscopy of microtubule dynamics revealed reduced depolymerization rates and increased stability. Mitotic exit signaled by breakdown of the mitotic spindle was delayed. In meiosis, microtubules displayed prolonged contact to the cell cortex during horsetail movement, followed by completion of meiosis I but frequent asymmetric failure of meiosis II spindle formation. Our results indicate that depolymerization dynamics modulated through interdomain motion may be important for regulating a subset of plus-end microtubule complexes in Schizosaccharomyces pombe.
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Ciclo Celular/fisiología , Meiosis/fisiología , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Fenilalanina/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Tirosina/metabolismoRESUMEN
Heme, a major functional form of iron in the cell, is synthesized in the mitochondria by ferrochelatase inserting ferrous iron into protoporphyrin IX. Heme deficiency was induced with N-methylprotoporphyrin IX, a selective inhibitor of ferrochelatase, in two human brain cell lines, SHSY5Y (neuroblastoma) and U373 (astrocytoma), as well as in rat primary hippocampal neurons. Heme deficiency in brain cells decreases mitochondrial complex IV, activates nitric oxide synthase, alters amyloid precursor protein, and corrupts iron and zinc homeostasis. The metabolic consequences resulting from heme deficiency seem similar to dysfunctional neurons in patients with Alzheimer's disease. Heme-deficient SHSY5Y or U373 cells die when induced to differentiate or to proliferate, respectively. The role of heme in these observations could result from its interaction with heme regulatory motifs in specific proteins or secondary to the compromised mitochondria. Common causes of heme deficiency include aging, deficiency of iron and vitamin B6, and exposure to toxic metals such as aluminum. Iron and B6 deficiencies are especially important because they are widespread, but they are also preventable with supplementation. Thus, heme deficiency or dysregulation may be an important and preventable component of the neurodegenerative process.