Abstract Body:

The endocytic system serves as the signal processing hub of the cell, ensuring molecular signals are targeted to the correct subcellular destination for the appropriate cell response. This requires an extraordinarily precise yet dynamic system to adapt to an ever-changing intra- and extra-cellular environment. Actin networks specific to these endocytic organelles have emerged as an essential component in ensuring proper function of these signal processing compartments [1]. The ubiquity of actin filaments within the cell necessitates a suite of molecules for spatiotemporal regulation of actin network formation. In this context, AnnexinA2 (AnxA2) associates with endosomal membranes, organizing membrane domains, and has been found to be essential for the formation of endosomal actin patches [2-4]. However, the molecular mechanism of AnxA2 regulation of actin mediated membrane rearrangements required of endocytic organelles remains unclear.

To date, imaging techniques have been limited either in spatial resolution or ability to unambiguously trace proteins and actin filaments on organelles. Combining precise fluorescence localization information from cryo-Structured Illumination Microscopy (cryoSIM) with high resolution ultrastructural information from cryo-Soft X-ray Tomography (cryoSXT) enables visualization of the 3D architecture of AnxA2 mediated interactions between actin networks and endosomal membranes at near physiological conditions (Fig. 1A). The enhanced penetration depth of X-rays allows for imaging of whole cells and absorption contrast eliminates the need for additional contrast enhancing treatment allowing imaging to 25nm resolution with minimal sample manipulation [5]. With such direct observation of the endocytic system, we set out to characterise how AnxA2 mediates endosomal system response to cellular stressors through its actin organisation abilities. By challenging cells to activate disparate signalling pathways, dynamic restructuring of endosomal membranes allowed us to track the distribution of AnxA2 and its regulation of the cytoskeleton network under different environmental conditions.

To visualize AnxA2, AnxA2-GFP was introduced into a U2OS cell line. Cells were cultured then plunge frozen, preserving cells in a near-native, fully hydrated state enabling visualization of the physiological cellular context. Fluorescence data corroborates successful fluorescent protein expression in addition to distinct AnxA2 puncta predominantly in the perinuclear region (Fig. 1B). Induction of inflammatory or hypoxic pathways reveals changes in mitochondrial morphology with mitochondria becoming enlarged, rounded, and showing increased signs of mitochondrial fission (Fig. 1B). AnxA2 puncta increases in abundance while fluorescence signal at the ultrastructure level reveals subtle changes in AnxA2 localization to different vesicular organelles and AnxA2 accumulation on structures not seen in untreated cells (Fig. 1C & D). AnxA2 function is context dependent and presumably repurposes actin to induce membrane remodelling in aid of cell responses to external stressors, potentially recruiting different subpopulations of endosomes in the stress response. Combined with a repertoire of organelle dyes, targeted ultrastructure visualization and AnxA2 localization relative to various cytoskeletal and endocytic components under different cellular contexts have been established.

Correlative Light and X-ray microscopy (CLXM) applies the advantages of two individually powerful techniques to the same sample and therefore, offers a unique view of the endocytic system. Redistribution patterns of AnxA2 and actin can be documented relative to corresponding ultrastructural changes in response to environmental challenges. This offers clues into which stage of actin network formation AnxA2 may be involved in i.e. recruitment, nucleation, stabilization, and/or anchoring the cytoskeleton to organelle membranes. Clarifying the roles of AnxA2 will provide a more comprehensive understanding of the molecular mechanisms governing localized actin network formation for endosomal biosynthesis and function. Insight into the regulatory mechanisms at endosomes and characterising this emerging role for actin will be essential in understanding the fundamental cellular infrastructure underlying cell signal processing, and how perturbations to this system contributes to aberrant cell signalling.

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