Background/Purpose: The synovium is primarily built by fibroblast-like-synoviocytes (FLS). These cells form a complex tissue network via long-distance connections (nanotubes) and wide intercellular matrix spaces. FLS coordinate and transfer information between each other to carry out tissue functions that are critical to joint homeostasis. For this, they exchange cargo, i.e. organelles like mitochondria either via transfer through interconnecting nanotubes or vesicles. Here we search for the underlying mechanisms of cell-to-cell cargo transfer. As targets, we selected actin and tubulin, both part of the cytoskeleton of nanotube structures, as well as the clathrin pathway for endocytosis.

Methods: Human FLS were taken from joint synovectomies. Passaged FLS were used to generate micromass cell cultures using Matrigel (BD®). Within just a few days these cells build a 3D structure that strongly resembles the in-vivo situation of the synovium. Cells were dyed with Celltracker and Mitotracker dyes (TF®) and challenged with various blocking agents. Analyses of the 3D confocal and multiphoton imaging data were done with Imaris Bitplane® software.

Results: By extracting the actin skeleton from the cells body, we show the triangular arrangement of actin filaments connected by the Arp2/3 complex at the basis of nanotubes as well as their linear arrangement in building nanotubes using electron microscopy. To block actin filament construction via the Arp2/3 complex we used CK666. This not only changed the architecture of the tissue but also significantly lowered the mitochondrial transfer rate between cells.

While thin nanotubes only consist of actin filaments, thick nanotubes may also contain microtubules. Additionally, kinesins use microtubules to shuttle cargo through the cytoplasm. To block tubulin synthesis, we used nocodazole. This treatment changed the structure of FLS micromasses, however, did not significantly lower the mitochondrial transfer rate. Moreover, we determined the mitochondrial travelling speed through nanotubes at 45,4 μm/h. This contrasts kinesin-mediated shuttling which amounts to 0,005 μm/h.

Clathrin mediated endocytosis, and thus the transfer of vesicles between cells, can be blocked by sucrose. Intriguingly, this treatment does not only affect vesicle transfer but also the formation of nanotubes, as it hinders the cell to build curvatures into its membrane. Sucrose treatment resulted in both a change in tissue architecture and significantly lowered the mitochondrial transfer rate.

Conclusion: Since the mitochondrial transfer rate was not affected by blocking the synthesis of microtubules and the traveling speed of mitochondria in our experiments was very different from that of kinesin shuttling, we conclude that microtubules are not involved in cell-to-cell cargo transfer. However, by blocking actin synthesis as well as clathrin mediated endocytosis, mitochondrial transfer was significantly lowered. Thus, cell-to-cell cargo transfer is likely an important feature of synovial tissue function that operates using distinct cellular pathways. Further studies will demonstrate its significance in the normal as well as the diseased synovium.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/in-search-of-mechanisms-underlying-fibroblast-like-synoviocyte-cell-to-cell-cargo-transfer/