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Cryo-Electron Tomography of Intracellular Condensates

Illustration of cryo-electron tomography of intracellular condensates in a vitrified eukaryotic cell.
Figure 1: This digital illustration demonstrates the core concept of cryo-electron tomography (cryo-ET) applied to intracellular condensates. The image showcases a vitrified eukaryotic cell section, depicting membraneless condensates within a cutaway side view. A tomographic electron beam is shown interacting with the hydrated specimen, highlighting the method's ability to reveal 3D structures while preserving hydration. Annotated labels identify key cellular components such as condensates, ribosomes, the endoplasmic reticulum, and regions prepared by cryo-FIB. The visual emphasizes how cryo-ET uniquely maintains native cellular conditions, using a cool color palette to reflect the cryogenic process.

Intracellular condensates, often referred to as membraneless organelles, are dynamic biomolecular assemblies that form through processes like liquid-liquid phase separation (LLPS). These compartments play crucial roles in organizing cellular biochemistry by concentrating specific proteins and nucleic acids, thereby facilitating diverse functions such as RNA metabolism, stress response, signaling, and DNA organization. Understanding their native structure, organization within the crowded cellular environment, and interactions with other organelles is paramount to deciphering their physiological roles and contributions to disease.

Cryo-electron tomography (cryo-ET) has emerged as a revolutionary imaging technique, uniquely capable of visualizing these delicate structures in their near-native, hydrated state within intact cells at macromolecular resolution. This article reviews how cryo-ET is transforming our comprehension of intracellular condensate architecture, the molecular mechanisms governing their formation, their functional implications, and how it fuels novel hypotheses about their orchestration of cellular life. Cryo-ET, particularly when coupled with correlative light and electron microscopy (CLEM) and advanced sample preparation techniques like cryo-focused ion beam (cryo-FIB) milling, allows researchers to pinpoint and dissect condensates within the complex 3D landscape of the cell. This approach bridges the gap between light microscopy observations of condensate dynamics and the high-resolution structural details that underpin their function, providing unprecedented insights into their in situ organization and material properties.

Unveiling Condensate Architecture in Native Cellular Landscapes

A primary strength of cryo-ET is its ability to capture the unadulterated morphology and spatial context of intracellular condensates. Studies using cryo-ET have revealed the intricate architectures of a wide array of these bodies. For instance, viral factories, which are sites of viral replication, have been shown by cryo-ET to be complex condensates recruiting host and viral components, with internal organization that supports viral assembly, as seen in mammalian reovirus (MRV) infections (Liu, X. et al., 2024). Similarly, the structure of stress granules and P-bodies, involved in mRNA triage and decay, are being elucidated in their cellular milieu, revealing their interactions with ribosomes and the endoplasmic reticulum.

Technological advancements are continually pushing the boundaries of what cryo-ET can achieve. Cryo-FIB milling sculpts vitrified cells into thin, electron-transparent lamellae suitable for tomography. Innovations like serialized on-grid lift-in sectioning for tomography (SOLIST) (Nguyen, H.T.D. et al., 2024) and Serial Lift-Out (Schiøtz, O.H. et al., 2023) are enhancing throughput and allowing for the examination of condensates in more complex samples like tissues and even small organisms. Furthermore, genetically encoded multimeric tags (GEMs) that are visible in both fluorescence microscopy and cryo-ET are improving the specific localization and identification of target proteins within condensates (Fung, H.K.H. et al., 2023).

Illustration of the process for imaging intracellular condensates using cryo-electron tomography, showing steps from cell to 3D reconstruction.
Figure 2: This illustration depicts the advancements in cryo-electron tomography (cryo-ET) used for imaging intracellular condensates. The process begins with a cross-section of an intact cell, followed by cryo-FIB milling that prepares thin vitrified sections, suitable for high-resolution imaging. Correlative Light and Electron Microscopy (CLEM) techniques are used to overlay fluorescence microscopy details on these sections, enhanced with SOLIST for increased resolution. Genetically encoded multimeric tags (GEMs) offer specificity by tagging points of interest. The image culminates in 3D reconstructed models of condensate structures where spatial registration is accurately transferred from fluorescence to electron microscopy, showcasing the synergistic use of these advanced techniques in structural biology.

These tools are crucial for visualizing how condensates, such as those involved in chromatin organization (Chen, J.K. et al., 2025; Uckelmann, M. et al., 2024) or polyphosphate storage (Chawla, R. et al., 2024), engage with other cellular structures like the cytoskeleton or organelle membranes.

Molecular Mechanisms of Condensate Assembly and Material States

Cryo-ET provides direct visual evidence for the molecular interactions that drive condensate formation and determine their material properties. The multivalency of protein interactions, often mediated by intrinsically disordered regions (IDRs) and specific motifs like phenylalanine-glycine (FG) repeats in nucleoporins (Ibáñez de Opakua, A. et al., 2024; Ng, S.C. et al., 2023), are key drivers of LLPS. Cryo-ET can visualize the resulting supramolecular assemblies, from loosely organized networks to more densely packed structures. For example, studies on Nup98 FG domains show how spacer length and motif identity dictate phase density and barrier properties, crucial for nuclear pore function.

The material state of condensates—ranging from highly dynamic liquids to more stable gels or even solid-like assemblies—is increasingly recognized as a critical functional parameter. Lasker, K. et al. (2022) demonstrated that the material properties of bacterial PopZ condensates, tunable by balancing attractive and repulsive forces, are critical for proper cell division. Cryo-ET, by capturing snapshots of these assemblies, can infer these properties from the packing density and organization of their constituents. Oh, H.J. et al. (2025) even used cryo-ET to visualize the 3D structure of size-controlled protein condensates stabilized by engineered interfacial protein cages, demonstrating how condensate properties can be externally modulated. These studies highlight how the specific molecular makeup and interaction network within a condensate, accessible by cryo-ET, dictate its physical nature and ultimately its biological activity.

3D molecular-level visualization of condensate assembly showing liquid-like and gel-like states through cryo-ET.
Figure 3: This 3D scientific illustration depicts the molecular-level visualization of condensate assembly as observed through cryo-electron tomography (cryo-ET). On the left side, it shows loosely organized, liquid-like condensates characterized by polymer-like networks of proteins with intrinsic disorder regions (IDRs) and FG-repeats, indicating flexibility and dynamic interactions. On the right, it illustrates densely packed, gel or solid-like condensates, featuring tightly organized structures due to strong multivalent protein interactions. The split-panel design highlights the structural differences captured in cryo-tomograms, with annotations explaining the supramolecular organization and the role of specific protein interactions in determining these material states.

Functional Roles and Dysregulation of Condensates Imaged by Cryo-ET

By linking observed structures to cellular activities, cryo-ET powerfully illuminates the functional roles of condensates. For instance, cryo-ET has shown how FMRP granules, implicated in Fragile X syndrome, are recruited to mitochondrial fission sites and locally translate proteins like MFF to regulate mitochondrial dynamics (Fenton, A.R. et al., 2024). In autophagy, phase separation of initiation hubs on cargo surfaces, visualized in yeast and human cells, acts as a trigger switch for selective autophagy (Licheva, M. et al., 2024). The pyrenoid, a CO2-fixing organelle in algae, utilizes matrix-traversing membranes whose biogenesis, involving proteins SAGA1 and MITH1, has been structurally characterized, revealing their role in creating adhesive interactions between membrane and matrix (Hennacy, J.H. et al., 2024).

The technique is also indispensable for understanding how viruses exploit or create condensates. HIV-1, for example, forms nuclear membraneless organelles (HIV-1 MLOs) that persist for weeks, shield the viral genome, and license reverse transcription, as visualized in vivo (Ay, S. et al., 2024). The HIV capsid itself has been shown to mimic karyopherin engagement of FG-nucleoporins to penetrate the nuclear pore condensate (Dickson, C.F. et al., 2023). Similarly, the proteomic analysis of SARS-CoV-2 particles has implicated host stress granule proteins like G3BP1/2 in viral assembly, with their roles likely linked to condensate formation (Murigneux, E. et al., 2024). Aberrant condensate behavior is also central to neurodegenerative diseases. Studies on C9orf72 dipeptide repeat proteins show their aggregation and interaction with molecular chaperones (Liu, F. et al., 2022), and cryo-ET can potentially visualize these pathogenic assemblies in situ.

3D scientific render of intracellular condensates in cellular contexts, showing FMRP granules, viral factories, stress granules, and neurodegenerative pathogenic condensates.
Figure 4: This 3D scientific render illustrates the diverse functional contexts of intracellular condensates as revealed by cryo-electron tomography (cryo-ET). It shows FMRP granules at mitochondrial fission sites, illustrating the interaction with dividing mitochondria, crucial for cell metabolism and energy distribution. Viral factories are depicted as organized assemblies engaged in the production of new virions, highlighting their role in viral replication within host cells. Stress granules are visualized interacting with the endoplasmic reticulum (ER), emphasizing their involvement in cellular stress responses and signaling pathways. Additionally, pathogenic protein condensates associated with neurodegenerative diseases are shown, indicating their disruptive distribution in neural cells and their impact on neuronal function. Each process is represented with spatial cues to display their cellular context accurately.

Generative Insights and Novel Hypotheses from Cryo-ET Studies

Beyond confirming existing models, cryo-ET is a hypothesis-generating engine. The direct visualization of condensate ultrastructure and interactions sparks new ideas about their regulation and function.

One emerging concept is the "condensate interactome". Cryo-ET reveals that condensates are not isolated entities but are often in close apposition to other organelles (e.g., ER, mitochondria) and even other condensates. We propose that this network of physical and functional interactions forms a higher-order regulatory layer, orchestrating complex cellular responses. For instance, the interface between a stress granule and the ER could facilitate localized translation or stress signaling in ways not possible if they were spatially segregated.

Secondly, the material properties of condensates may function as a dynamic, programmable code. The transition between liquid-like, gel-like, and solid-like states, visualized by differences in molecular packing by cryo-ET (Lasker, K. et al., 2022; Uckelmann, M. et al., 2024), could be actively tuned by the cell to gate molecular access, control reaction kinetics, or alter mechanical properties in response to stimuli. This "material code" might be as important as sequence-specific binding for regulating condensate function.

Thirdly, the phenomenon of viral mimicry of host condensate components, as seen with the HIV capsid and FG-nucleoporins (Dickson, C.F. et al., 2023), might be a widespread viral strategy. We hypothesize that other viruses may have evolved mechanisms to structurally or dynamically mimic components of host condensates, allowing them to co-opt these structures, disrupt their antiviral functions, or use them as havens for replication, camouflaged from immune detection. Cryo-ET is perfectly poised to identify such molecular mimicry at the virus-host condensate interface.

Finally, the internal architecture of condensates, often revealed by cryo-ET to be heterogeneous rather than uniform (Liu, X. et al., 2024), suggests sophisticated internal organization for information processing. Sub-compartments, molecular gradients, or defined interfaces with structures like microtubules could serve as platforms for sequential enzymatic reactions, assembly lines for macromolecular complexes, or channels for vectorial transport of molecules through the condensate, far exceeding simple concentration effects.

Illustration of generative hypotheses from cryo-ET depicting condensate interactome, tunable material code transitions, viral mimicry, and heterogeneous organization.
Figure 5: This conceptual illustration explains the generative hypotheses arising from cryo-electron tomography (cryo-ET) studies. It highlights the condensate interactome, showing networks that connect cellular condensates to organelles, illustrating the dynamic relationships that form a complex interaction network within the cellular environment. The tunable material code is depicted through transitions among liquid, gel, and solid states of these condensates, emphasizing their adaptable nature. Viral mimicry is represented by interfaces where viral structures resemble host condensates, indicating potential pathways for viral hijacking of cellular processes. Lastly, the diagram includes heterogeneous internal organization within condensates, illustrating their varied structural complexity. Together, these elements portray higher-order cellular regulation and information processing, showing how these diverse layers contribute to cellular functionality.

Conclusion

Cryo-electron tomography is fundamentally reshaping our understanding of intracellular condensates, providing unparalleled insights into their native architecture, molecular assembly, material properties, and functional integration within the cellular ecosystem. The ability to visualize these dynamic, often transient structures in their unperturbed state is bridging cellular and molecular biology, allowing us to witness biological processes at an unprecedented level of detail.

The ongoing development of cryo-ET methodologies, including improved sample preparation, faster data acquisition, and sophisticated image processing, promises even greater resolving power and the ability to capture dynamic events. Future challenges include imaging rarer or smaller condensates, achieving higher in-cell resolutions to discern individual protein conformations and interactions, and integrating cryo-ET data with complementary 'omics' and live-cell imaging approaches for a truly holistic view. Open questions abound: What is the full complement of condensates within a given cell type? What are the precise rules governing their spatiotemporal assembly and disassembly? How do alterations in condensate structure and dynamics contribute to the full spectrum of human diseases? Cryo-ET will undoubtedly be at the forefront of addressing these questions, continuing to drive conceptual breakthroughs and revealing the intricate, condensed world within our cells.

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