Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
J Cell Sci ; 136(4)2023 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-36790431

RESUMEN

In 1891, the existence of an X chromosome was noted for the first time. Hermann Henking was studying spermatocyte divisions of the firebug Pyrrhocoris apterus and observed that one chromosome behaved differently than all of the rest of the chromosomes. Henking called this chromosome 'Element x'. Henking's discovery of the X element (later called X chromosome) initiated more than a century of fascinating genetics and cell biology, forming the foundation of several avenues of research in biology. His work led to exploration of a number of questions in a wide range of model systems and very soon to the abandonment of the firebug as a model for studies on the behavior of chromosomes in meiosis. Here, we argue that studies on both bivalent and univalent chromosome behavior in general, and work on how to solve chromosome lagging to prevent aneuploidy in particular, should lead us back to using the firebug as a model for error correction during cell division.


Asunto(s)
Cromosomas , Meiosis , Humanos , Cromosomas/genética , Aneuploidia
2.
J Microsc ; 284(1): 25-44, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34110027

RESUMEN

We present a software-assisted workflow for the alignment and matching of filamentous structures across a three-dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labour. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite dealing with imperfect data related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time-consuming. Furthermore, an interactive visualisation of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialised, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualisation and interaction tools for each processing step to guarantee real-time response, and an optimised workflow that efficiently guides the user through datasets. The final solution presented here is the result of an iterative process with tight feedback loops between the involved computer scientists and cell biologists. LAY DESCRIPTION: Electron tomography of biological samples is used for a three-dimensional (3D) reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large-scale electron tomography can be applied to increase the reconstructed volume for the visualisation of full spindles. For this, each spindle is cut into a series of semi-thick physical sections, from which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (ie physical distortions) caused by the image acquisition process, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi-automatic solution that considers all requirements for large-scale stitching of microtubules in image stacks of consecutive sections. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large-scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts.


Asunto(s)
Tomografía con Microscopio Electrónico , Huso Acromático , Tomografía/instrumentación , Técnicas Histológicas , Procesamiento de Imagen Asistido por Computador/instrumentación , Imagenología Tridimensional , Microtúbulos , Programas Informáticos
3.
Development ; 144(18): 3253-3263, 2017 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-28827395

RESUMEN

Asymmetric partitioning is an essential component of many developmental processes. As spermatogenesis concludes, sperm are streamlined by discarding unnecessary cellular components into cellular wastebags called residual bodies (RBs). During nematode spermatogenesis, this asymmetric partitioning event occurs shortly after anaphase II, and both microtubules and actin partition into a central RB. Here, we use fluorescence and transmission electron microscopy to elucidate and compare the intermediate steps of RB formation in Caenorhabditis elegans, Rhabditis sp. SB347 (recently named Auanema rhodensis) and related nematodes. In all cases, intact microtubules reorganize and move from centrosomal to non-centrosomal sites at the RB-sperm boundary whereas actin reorganizes through cortical ring expansion and clearance from the poles. However, in species with tiny spermatocytes, these cytoskeletal changes are restricted to one pole. Consequently, partitioning yields one functional sperm with the X-bearing chromosome complement and an RB with the other chromosome set. Unipolar partitioning may not require an unpaired X, as it also occurs in XX spermatocytes. Instead, constraints related to spermatocyte downsizing may have contributed to the evolution of a sperm cell equivalent to female polar bodies.


Asunto(s)
División Celular Asimétrica , Caenorhabditis elegans/citología , Tamaño de la Célula , Citoesqueleto/metabolismo , Razón de Masculinidad , Espermatozoides/citología , Actinas/metabolismo , Animales , Centrosoma/metabolismo , Citoesqueleto/ultraestructura , Femenino , Organismos Hermafroditas/citología , Masculino , Meiosis , Microtúbulos/metabolismo , Modelos Biológicos , Espermatocitos/citología , Espermatocitos/ultraestructura , Espermatogénesis , Espermatozoides/ultraestructura
5.
Chromosoma ; 125(2): 277-86, 2016 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26511278

RESUMEN

In males of many taxa, univalent sex chromosomes normally segregate during the first meiotic division, and analysis of sex chromosome segregation was foundational for the chromosome theory of inheritance. Correct segregation of single or multiple univalent sex chromosomes occurs in a cellular environment where every other chromosome is a bivalent that is being partitioned into homologous chromosomes at anaphase I. The mechanics of univalent chromosome segregation vary among animal taxa. In some, univalents establish syntelic attachment of sister kinetochores to the spindle. In others, amphitelic attachment is established. Here, we review how this problem of segregation of unpaired chromosomes is solved in different animal systems. In addition, we give a short outlook of how mechanistic insights into this process could be gained by explicitly studying model organisms, such as Caenorhabditis elegans.


Asunto(s)
Segregación Cromosómica , Meiosis , Cromosomas Sexuales/genética , Animales , Femenino , Humanos , Cinetocoros/metabolismo , Masculino , Cromosomas Sexuales/metabolismo
6.
Methods Cell Biol ; 187: 1-41, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38705621

RESUMEN

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.


Asunto(s)
Microscopía Electrónica de Transmisión , Humanos , Microscopía Electrónica de Transmisión/métodos , Animales , Microscopía por Crioelectrón/métodos , Microscopía Electrónica/métodos , Microscopía Inmunoelectrónica/métodos , Microscopía Fluorescente/métodos , Substitución por Congelación/métodos
7.
Nat Protoc ; 19(5): 1436-1466, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38424188

RESUMEN

Volume electron microscopy is the method of choice for the in situ interrogation of cellular ultrastructure at the nanometer scale, and with the increase in large raw image datasets generated, improving computational strategies for image segmentation and spatial analysis is necessary. Here we describe a practical and annotation-efficient pipeline for organelle-specific segmentation, spatial analysis and visualization of large volume electron microscopy datasets using freely available, user-friendly software tools that can be run on a single standard workstation. The procedures are aimed at researchers in the life sciences with modest computational expertise, who use volume electron microscopy and need to generate three-dimensional (3D) segmentation labels for different types of cell organelles while minimizing manual annotation efforts, to analyze the spatial interactions between organelle instances and to visualize the 3D segmentation results. We provide detailed guidelines for choosing well-suited segmentation tools for specific cell organelles, and to bridge compatibility issues between freely available open-source tools, we distribute the critical steps as easily installable Album solutions for deep learning segmentation, spatial analysis and 3D rendering. Our detailed description can serve as a reference for similar projects requiring particular strategies for single- or multiple-organelle analysis, which can be achieved with computational resources commonly available to single-user setups.


Asunto(s)
Imagenología Tridimensional , Microscopía Electrónica , Programas Informáticos , Microscopía Electrónica/métodos , Imagenología Tridimensional/métodos , Orgánulos/ultraestructura , Análisis Espacial , Procesamiento de Imagen Asistido por Computador/métodos , Humanos , Microscopía Electrónica de Volumen
8.
Elife ; 122024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-39102289

RESUMEN

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.


Asunto(s)
Barrera Hematoencefálica , Tomografía con Microscopio Electrónico , Animales , Ratones , Tomografía con Microscopio Electrónico/métodos , Barrera Hematoencefálica/ultraestructura , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/ultraestructura , Ratones Endogámicos C57BL , Masculino , Microscopía Electrónica de Rastreo/métodos , Microtomía
9.
Circ Heart Fail ; 17(6): e011107, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38847102

RESUMEN

BACKGROUND: Clinical studies demonstrated beneficial effects of sodium-glucose-transporter 2 inhibitors on the risk of cardiovascular death in patients with heart failure with preserved ejection fraction (HFpEF). However, underlying processes for cardioprotection remain unclear. The present study focused on the impact of empagliflozin (Empa) on myocardial function in a rat model with established HFpEF and analyzed underlying molecular mechanisms. METHODS: Obese ZSF1 (Zucker fatty and spontaneously hypertensive) rats were randomized to standard care (HFpEF, n=18) or Empa (HFpEF/Empa, n=18). ZSF1 lean rats (con, n=18) served as healthy controls. Echocardiography was performed at baseline and after 4 and 8 weeks, respectively. After 8 weeks of treatment, hemodynamics were measured invasively, mitochondrial function was assessed and myocardial tissue was collected for either molecular and histological analyses or transmission electron microscopy. RESULTS: In HFpEF Empa significantly improved diastolic function (E/é: con: 17.5±2.8; HFpEF: 24.4±4.6; P<0.001 versus con; HFpEF/Empa: 19.4±3.2; P<0.001 versus HFpEF). This was accompanied by improved hemodynamics and calcium handling and by reduced inflammation, hypertrophy, and fibrosis. Proteomic analysis demonstrated major changes in proteins involved in mitochondrial oxidative phosphorylation. Cardiac mitochondrial respiration was significantly impaired in HFpEF but restored by Empa (Vmax complex IV: con: 0.18±0.07 mmol O2/s/mg; HFpEF: 0.13±0.05 mmol O2/s/mg; P<0.041 versus con; HFpEF/Empa: 0.21±0.05 mmol O2/s/mg; P=0.012 versus HFpEF) without alterations of mitochondrial content. The expression of cardiolipin, an essential stability/functionality-mediating phospholipid of the respiratory chain, was significantly decreased in HFpEF but reverted by Empa (con: 15.9±1.7 nmol/mg protein; HFpEF: 12.5±1.8 nmol/mg protein; P=0.002 versus con; HFpEF/Empa: 14.5±1.8 nmol/mg protein; P=0.03 versus HFpEF). Transmission electron microscopy revealed a reduced size of mitochondria in HFpEF, which was restored by Empa. CONCLUSIONS: The study demonstrates beneficial effects of Empa on diastolic function, hemodynamics, inflammation, and cardiac remodeling in a rat model of HFpEF. These effects were mediated by improved mitochondrial respiratory capacity due to modulated cardiolipin and improved calcium handling.


Asunto(s)
Compuestos de Bencidrilo , Diástole , Modelos Animales de Enfermedad , Glucósidos , Insuficiencia Cardíaca , Mitocondrias Cardíacas , Ratas Zucker , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Volumen Sistólico , Animales , Glucósidos/farmacología , Compuestos de Bencidrilo/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Cardíaca/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Diástole/efectos de los fármacos , Volumen Sistólico/efectos de los fármacos , Masculino , Función Ventricular Izquierda/efectos de los fármacos , Ratas Endogámicas SHR , Transporte de Electrón/efectos de los fármacos , Ratas
10.
Bio Protoc ; 13(20): e4849, 2023 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-37900106

RESUMEN

For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/). Key features • High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM). • Serial-section electron tomography of selected cells. • Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization.

11.
Curr Biol ; 33(5): 791-806.e7, 2023 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-36693370

RESUMEN

Centrosomes are cellular structures that nucleate microtubules. At their core is a pair of centrioles that recruit pericentriolar material (PCM). Although centrosomes are considered membraneless organelles, in many cell types, including human cells, centrosomes are surrounded by endoplasmic reticulum (ER)-derived membranes of unknown structure and function. Using volume electron microscopy (vEM), we show that centrosomes in the Caenorhabditis elegans (C. elegans) early embryo are surrounded by a three-dimensional (3D), ER-derived membrane reticulum that we call the centriculum, for centrosome-associated membrane reticulum. The centriculum is adjacent to the nuclear envelope in interphase and early mitosis and fuses with the fenestrated nuclear membrane at metaphase. Centriculum formation is dependent on the presence of an underlying centrosome and on microtubules. Conversely, increasing centriculum size by genetic means led to the expansion of the PCM, increased microtubule nucleation capacity, and altered spindle width. The effect of the centriculum on centrosome function suggests that in the C. elegans early embryo, the centrosome is not membraneless. Rather, it is encased in a membrane meshwork that affects its properties. We provide evidence that the centriculum serves as a microtubule "filter," preventing the elongation of a subset of microtubules past the centriculum. Finally, we propose that the fusion between the centriculum and the nuclear membrane contributes to nuclear envelope breakdown by coupling spindle elongation to nuclear membrane fenestration.


Asunto(s)
Caenorhabditis elegans , Centrosoma , Animales , Humanos , Caenorhabditis elegans/genética , Centrosoma/metabolismo , Centriolos/metabolismo , Microtúbulos/metabolismo , Mitosis , Estrés del Retículo Endoplásmico
12.
PLoS One ; 18(7): e0285073, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37498864

RESUMEN

Spermatogenesis leads to the formation of functional sperm cells. Here we have applied high-pressure freezing in combination with transmission electron microscopy (TEM) to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea, a species in which spermatogenesis had not previously been studied. We show the ultrastructure of different stages of sperm development in this species. Thorough examination of TEM data and electron tomographic reconstructions revealed interesting structural features of the nebenkern, an organelle composed of fused mitochondria that has been studied in spermatids of other insect species. We have applied serial-section electron tomography of the nebenkern to demonstrate in three dimensions (3D) that this organelle in H. membranacea is composed of two interwoven mitochondrial derivatives, and that the mitochondrial derivatives are connected by a zipper-like structure at opposing positions. Our approach will enable further ultrastructural analyses of the nebenkern in other organisms.


Asunto(s)
Mantódeos , Animales , Masculino , Semen , Espermatogénesis , Espermatozoides , Espermátides , Mitocondrias
13.
Elife ; 112022 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-35894209

RESUMEN

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the chromosomes and the rest of the spindle. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here, we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers and their surrounding MTs in detail. We found that k-fibers exhibit remarkable variation in circumference and KMT density along their length, with the pole-proximal side showing a broadening. Extending our structural analysis then to other MTs in the spindle, we further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for KMT growth and k-fiber self-organization models as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2022). Finally, we also introduce a new visualization tool allowing an interactive display of our 3D spindle data that will serve as a resource for further structural studies on mitosis in human cells.


Asunto(s)
Cinetocoros , Huso Acromático , Animales , Cromosomas , Células HeLa , Humanos , Mamíferos , Metafase , Microtúbulos/ultraestructura , Huso Acromático/ultraestructura
14.
PLoS One ; 17(8): e0272978, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35960713

RESUMEN

Praying mantids are important models for studying a wide range of chromosome behaviors, yet few species of mantids have been characterized chromosomally. Here we show that the praying mantid Hierodula membranacea has a chromosome number of 2n = 27, and X1X1X2X2 (female): X1X2Y (male) sex determination. In male meiosis I, the X1, X2, and Y chromosomes of H. membranacea form a sex trivalent, with the Y chromosome associating with one spindle pole and the X1 and X2 chromosomes facing the opposite spindle pole. While it is possible that such a sex trivalent could experience different spindle forces on each side of the trivalent, in H. membranacea the sex trivalent aligns at the spindle equator with all of the autosomes, and then the sex chromosomes separate in anaphase I simultaneously with the autosomes. With this observation, H. membranacea can be used as a model system to study the balance of forces acting on a trivalent during meiosis I and analyze the functional importance of chromosome alignment in metaphase as a preparatory step for subsequent correct chromosome segregation.


Asunto(s)
Mantódeos , Animales , Segregación Cromosómica , Femenino , Masculino , Mantódeos/genética , Meiosis/genética , Metafase , Cromosomas Sexuales , Huso Acromático , Cromosoma Y
15.
Elife ; 112022 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-35876665

RESUMEN

During eukaryotic cell division, chromosomes are linked to microtubules (MTs) in the spindle by a macromolecular complex called the kinetochore. The bound kinetochore microtubules (KMTs) are crucial to ensuring accurate chromosome segregation. Recent reconstructions by electron tomography (Kiewisz et al., 2022) captured the positions and configurations of every MT in human mitotic spindles, revealing that roughly half the KMTs in these spindles do not reach the pole. Here, we investigate the processes that give rise to this distribution of KMTs using a combination of analysis of large-scale electron tomography, photoconversion experiments, quantitative polarized light microscopy, and biophysical modeling. Our results indicate that in metaphase, KMTs grow away from the kinetochores along well-defined trajectories, with the speed of the KMT minus ends continually decreasing as the minus ends approach the pole, implying that longer KMTs grow more slowly than shorter KMTs. The locations of KMT minus ends, and the turnover and movements of tubulin in KMTs, are consistent with models in which KMTs predominately nucleate de novo at kinetochores in metaphase and are inconsistent with substantial numbers of non-KMTs being recruited to the kinetochore in metaphase. Taken together, this work leads to a mathematical model of the self-organization of kinetochore-fibers in human mitotic spindles.


Asunto(s)
Cinetocoros , Huso Acromático , Segregación Cromosómica , Cromosomas , Humanos , Metafase , Microtúbulos/metabolismo , Mitosis , Huso Acromático/metabolismo
16.
Methods Cell Biol ; 162: 151-170, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33707011

RESUMEN

We introduce a new workflow that allows screening and selection of staged mammalian cells in mitosis prior to subsequent electron microscopy. We mainly describe four improved steps of specimen preparation. Firstly, we describe a method to efficiently enrich mammalian cells and attach them to sapphire discs; secondly, we report on the use of 3D-printed containers to seed cells on coated sapphire discs for high-pressure freezing; thirdly, we take advantage of a specimen carrier that allows for an upside-down placing of sapphire discs without a second carrier or spacer ring to close the "sandwich"; and fourthly, we use histological dyes to stain DNA/chromatin during freeze-substitution. Out of 14 tested histological dyes, we routinely use four of them for visual inspection of mitotic cells by light microscopy. Applying this streamlined workflow, HeLa cells at different stages of mitosis can be selected for further ultrastructural analysis. The practical aspects of this approach will be discussed herein.


Asunto(s)
Colorantes , Ensayos Analíticos de Alto Rendimiento , Animales , Substitución por Congelación , Células HeLa , Humanos , Microscopía Electrónica
17.
Bio Protoc ; 10(20): e3785, 2020 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-33659440

RESUMEN

Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for in situ filming of spermatocyte meiosis in whole immobilized C. elegans males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe the workflow for the acquisition and processing of data to obtain quantitative information about the dynamics of chromosome segregation in spermatocyte meiosis I and II. In addition, our newly developed approach allows us to re-orient filmed spindles in silico, regardless of the initial 3D orientation in the worm, and analyze spindle dynamics in living worms in a statistically robust manner. Our live-imaging approach is also applicable to C. elegans hermaphrodites and should be expandable to other fluorescently labelled nematodes or other fully transparent small model organisms.

18.
Elife ; 92020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32149606

RESUMEN

Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a 'tug of war' reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.


Asunto(s)
Emparejamiento Cromosómico/fisiología , Segregación Cromosómica/fisiología , Meiosis/fisiología , Espermatocitos/fisiología , Huso Acromático/fisiología , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Masculino , Cromosoma X
19.
Elife ; 92020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32966209

RESUMEN

The spindle shows remarkable diversity, and changes in an integrated fashion, as cells vary over evolution. Here, we provide a mechanistic explanation for variations in the first mitotic spindle in nematodes. We used a combination of quantitative genetics and biophysics to rule out broad classes of models of the regulation of spindle length and dynamics, and to establish the importance of a balance of cortical pulling forces acting in different directions. These experiments led us to construct a model of cortical pulling forces in which the stoichiometric interactions of microtubules and force generators (each force generator can bind only one microtubule), is key to explaining the dynamics of spindle positioning and elongation, and spindle final length and scaling with cell size. This model accounts for variations in all the spindle traits we studied here, both within species and across nematode species spanning over 100 million years of evolution.


Asunto(s)
Caenorhabditis elegans , Tamaño de la Célula , Microtúbulos , Huso Acromático , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Evolución Molecular , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Modelos Biológicos , Fenotipo , Huso Acromático/química , Huso Acromático/genética , Huso Acromático/metabolismo
20.
Methods Cell Biol ; 152: 119-134, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31326018

RESUMEN

We describe a routine method to locate cells of appropriate meiotic stages in the gonad of Caenorhabditis elegans males prior to 3D reconstruction of meiotic spindles by electron tomography. For this, serial semi-thick (300nm) sections of whole worms are pre-screened and recorded at low magnification by transmission electron microscopy. Cells of interest are identified in aligned image stacks showing the entire proximal region of male gonads at low magnification. Tilt series of selected cells are then recorded at higher magnification to reconstruct meiotic spindles of selected cells in 3D. Our approach allows a routine staging of spermatocytes without the use of anesthetics or the application of physical immobilization of worms. We also describe a modification of a previously published protocol (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007) by using polyvinylpyrrolidone (PVP) instead of bovine serum albumin (BSA) as a "filler" for specimen loading in high-pressure freezing.


Asunto(s)
Caenorhabditis elegans/fisiología , Meiosis/fisiología , Animales , Masculino , Microscopía Electrónica de Transmisión/métodos , Espermatocitos/fisiología , Huso Acromático/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA