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Abstract: Immune effects of membrane attack complexes (MAC) have been widely attributed to their abilities to cause cell death. Here, we show that the MAC component, C9, forms non-cytolytic aggregates with pro-inflammatory effects. Intracellular aggregates of C9 are detected within inflamed tissues of patients in association with endothelial cell (EC) activation but not increased cell death. We identify NUMBL as a Rab35 effector that directly binds surface-bound C9 to promote C9 internalization and entry into the endolysosomal pathway. Within acidified endolysosomes, C9 forms insoluble aggregates that are targeted for degradative aggrephagy in a process that activates NF-κB. For C9 aggrephagy to occur, ZFYVE21, a Rab5 effector, complexes with RNF34 to bridge C9 aggregates to LC3B+ aggresome membranes. We detect C9 aggregates in vivo, and we show that a ZFYVE21-RNF34 signaling axis is required for C9 aggrephagy and NF-κB -dependent EC activation in three separate mouse models. Mice with conditional loss of ZFYVE21 in ECs show reduced aggregraphy, resulting in attenuated systemic inflammation and reduced tissue injury following skin transplantation. Our data show that the C9 component of MACs forms intracellular aggregates with alarmin-like properties.

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Abstract: Understanding the intracellular fate of nanoparticles (NPs) is essential for advancing nanomedicine, particularly in targeted drug delivery for cancer therapy. Here, we present a complementary cryogenic microscopy workflow across scales to investigate the uptake and subcellular localization of zirconyl-containing inorganic–organic hybrid nanoparticles (IOH-NPs) in murine breast cancer cells. Our approach integrates cryogenic fluorescence microscopy (cryo-FM), cryo-focused ion beam scanning electron microscopy (cryo-FIBSEM), and cryo-soft X-ray tomography (cryo-SXT), enabling molecular specificity, high-resolution imaging, and volumetric ultrastructural analysis in near-native cellular states. We demonstrate that the cryogenic workflow provides enough contrast and resolution across all modalities for quantifying the IOH-NP uptake: NPs are internalized within 2 h of incubation and progressively accumulate in endolysosomes over time, as confirmed by fluorescence labeling and SXT. Quantitative analysis reveals a marked increase in endolysosomal accumulation of IOH-NPs from 2 to 24 h. Our findings help to establish multimodal cryogenic microscopy as a powerful tool for nanoscale imaging and quantitative analysis of NP uptake within close-to-native cells, offering new insights into NP trafficking and cellular responses relevant to nanomedicine development.

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Abstract: Upon exposure to biological environments, nanoparticles are rapidly coated with biomolecules, predominantly proteins, which alter their colloidal stability, biodistribution, and cell interactions. Despite extensive efforts to investigate the nanoparticles' fate, only a few studies use high-resolution characterization methods that allow in-depth characterization, and the existing methodologies are unable to differentiate particles internalized at the onset of incubation from those taken up toward the end of an incubation period. In this study, these limitations related to incubation disparities are overcame and precisely monitored the spatiotemporal displacement of colloidally stable protein corona-coated nanoparticles within cells. An unprecedented application of cryogenic X-ray nanotomography, combined with high-resolution, super-resolution, and correlative microscopy techniques, revealed the migration of nanoparticles to the perinuclear region while monitoring the evolution of cellular organelles in fully hydrated cells under near-native conditions, without the need for contrasting agents. Notably, this tracking indicates the progressive fusion of vesicles carrying the nanoparticles intracellularly. This strategy demonstrates the potential for uncovering the temporal aspects of nanoparticle behavior within cells and can be adaptable to a wide range of nanoparticles and cell types, offering a versatile and powerful tool to follow nanoparticles in cellular environments.

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Abstract: For decades, when scientists have wanted to study cell architecture in granular detail and to study how viruses assemble inside the cell, they've turned to transmission electron microscopy (TEM), a high-resolution imaging technique. However, TEM studies are limited to 2D sections and offer no information regarding the 3D geometry of different features. They also look at thin slivers rather than a whole cell, and slow throughput means TEM studies are conducted on a small number of samples. On Diamond’s B24 beamline, researchers can perform correlative fluorescence microscopy and X-ray tomography, imaging proteins using structured illumination microscopy under cryogenic conditions (cryoSIM) and capturing cellular ultrastructure from the same cells using cryo-soft-X-ray tomography (cryoSXT). In a preprint recently published on bioRxiv, an international team of researchers used this correlative light X-ray tomography (CLXT) approach to study the roles of nine genes in virus assembly in herpes simplex virus-1 (HSV-1). This multi-modal imaging strategy allowed a 3D study of viral assembly, highlighting the contributions that key HSV-1 proteins make to virus assembly and underscoring the power of correlative fluorescence and X-ray tomography cryo-imaging for studies of this type.

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Abstract: The influenza A virus (IAV) genome that consists of eight viral RNA (vRNA) segments is transcribed and replicated by the viral heterotrimeric RNA-dependent RNA polymerase (RdRP) complex, composed of polymerase acidic (PA), polymerase basic 1 (PB1), and PB2, subunits, in the cell nucleus. Replicated vRNA segments are assembled with RdRP and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packed into progeny virions. The GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this D.Phil. project, I have utilised a combination of biochemical and structural approaches to characterise the interaction between the IAV RdRP and Rab11a. Initially, using a GST pull-down assay, I showed that the interaction between the IAV RdRP and Rab11a is direct, and that the 627- domain of PB2 is required for this interaction. Furthermore, my study demonstrated for the first time that Rab11a has a differential specificity for vRNP but not cRNP. Analysis of the Rab11a interactome further revealed that IAV infection reduces Rab11a binding to Rab11-family interacting partners (Rab11-FIPs), thereby altering the stoichiometry of cytoplasmic motor proteins bound by Rab11a. Using mutant Rab11a in a GST pull-down assay, this study provides the first evidence that IAV RdRP binds to the same site on Rab11a as Rab11-FIPs. It was also investigated here whether Rab11a has a role in determining the host specificity of influenza viruses. However, this study could not identify any adaptive mutations in PB2 that may affect the interaction between RdRP and Rab11a. Moreover, the study also did not identify the exact site of Rab11a binding on the viral RdRP using cryo- electron microscopy. However, performing a small-angle X-ray scattering analysis I showed that C-terminal two thirds of PB2 (PB2-C) and Rab11a form a well-folded protein complex in solution, and confirmed in a controlled pull-down assay that the interaction between PB2-C and Rab11a is direct. Finally, I have also established a workflow to study the interaction between IAV vRNPs and Rab11a in the context of live infected cells using full field cryo-X-ray microscopy. The findings of my D.Phil. work expand our understanding of the cytoplasmic transport of the influenza virus vRNPs. Identifying the exact site of Rab11a binding on the viral RdRP could uncover a novel target site for the development of an influenza antiviral drug.