RESUMO
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
Assuntos
Citoplasma , Polirribossomos , Ribonucleoproteínas , Polirribossomos/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulação de Dinâmica Molecular , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Condensados Biomoleculares/metabolismo , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genéticaRESUMO
Most life scientists would agree that understanding how cellular processes work requires structural knowledge about the macromolecules involved. For example, deciphering the double-helical nature of DNA revealed essential aspects of how genetic information is stored, copied and repaired. Yet, being reductionist in nature, structural biology requires the purification of large amounts of macromolecules, often trimmed off larger functional units. The advent of cryogenic electron microscopy (cryo-EM) greatly facilitated the study of large, functional complexes and generally of samples that are hard to express, purify and/or crystallize. Nevertheless, cryo-EM still requires purification and thus visualization outside of the natural context in which macromolecules operate and coexist. Conversely, cell biologists have been imaging cells using a number of fast-evolving techniques that keep expanding their spatial and temporal reach, but always far from the resolution at which chemistry can be understood. Thus, structural and cell biology provide complementary, yet unconnected visions of the inner workings of cells. Here we discuss how the interplay between cryo-EM and cryo-electron tomography, as a connecting bridge to visualize macromolecules in situ, holds great promise to create comprehensive structural depictions of macromolecules as they interact in complex mixtures or, ultimately, inside the cell itself.
Assuntos
Biologia Celular , Células , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microscopia Crioeletrônica/métodos , Microscopia Crioeletrônica/tendências , Tomografia com Microscopia Eletrônica/métodos , Tomografia com Microscopia Eletrônica/tendências , Substâncias Macromoleculares/análise , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/ultraestrutura , Biologia Celular/instrumentação , Células/química , Células/citologia , Células/metabolismo , Células/ultraestrutura , HumanosRESUMO
Cellular cryo-electron tomography (cryo-ET) has emerged as a key method to unravel the spatial and structural complexity of cells in their near-native state at unprecedented molecular resolution. To enable quantitative analysis of the complex shapes and morphologies of lipid membranes, the noisy three-dimensional (3D) volumes must be segmented. Despite recent advances, this task often requires considerable user intervention to curate the resulting segmentations. Here, we present ColabSeg, a Python-based tool for processing, visualizing, editing, and fitting membrane segmentations from cryo-ET data for downstream analysis. ColabSeg makes many well-established algorithms for point-cloud processing easily available to the broad community of structural biologists for applications in cryo-ET through its graphical user interface (GUI). We demonstrate the usefulness of the tool with a range of use cases and biological examples. Finally, for a large Mycoplasma pneumoniae dataset of 50 tomograms, we show how ColabSeg enables high-throughput membrane segmentation, which can be used as valuable training data for fully automated convolutional neural network (CNN)-based segmentation.
Assuntos
Algoritmos , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Software , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Membrana Celular/ultraestrutura , Mycoplasma pneumoniae/ultraestrutura , Interface Usuário-Computador , Imageamento Tridimensional/métodosRESUMO
Helical filaments are essential macromolecular elements in cellular organization and dynamics. Recent advances in cryo-electron tomography allow faithful imaging of isolated or in-cell filaments. Here, we present a protocol to generate density maps at sub-nanometer resolution of helical filaments by subtomogram averaging, exemplified with isolated mumps virus nucleocapsids and their in-cell form as an extension of the protocol. We detail procedures from pre-processing of tilt-series movie frames to refinement of reconstructed averages for streamlined data processing of helical filaments. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.
RESUMO
Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near-atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD-related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology.
Assuntos
Gotículas Lipídicas , Microscopia Eletrônica , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Gotículas Lipídicas/química , Humanos , Animais , Microscopia Eletrônica/métodos , Metabolismo dos LipídeosRESUMO
Centrosomes organize microtubules that are essential for mitotic divisions in animal cells. They consist of centrioles surrounded by Pericentriolar Material (PCM). Questions related to mechanisms of centriole assembly, PCM organization, and microtubule formation remain unanswered, in part due to limited availability of molecular-resolution structural analyses in situ. Here, we use cryo-electron tomography to visualize centrosomes across the cell cycle in cells isolated from C. elegans embryos. We describe a pseudo-timeline of centriole assembly and identify distinct structural features including a cartwheel in daughter centrioles, and incomplete microtubule doublets surrounded by a star-shaped density in mother centrioles. We find that centriole and PCM microtubules differ in protofilament number (13 versus 11) indicating distinct nucleation mechanisms. This difference could be explained by atypical γ-tubulin ring complexes with 11-fold symmetry identified at the minus ends of short PCM microtubules. We further characterize a porous and disordered network that forms the interconnected PCM. Thus, our work builds a three-dimensional structural atlas that helps explain how centrosomes assemble, grow, and achieve function.