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Tunneling nanotube formation is driven by Eps8/IRSp53-dependent linear actin polymerization

ABSTRACT: Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that tunneling nanotubes formed through the outward extension of actin achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of tunneling nanotubes showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunneling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunneling nanotube formation.

SUBMITTER: J., Michael Henderson

PROVIDER: S-SCDT-10_15252-EMBJ_2023113761 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Tunneling nanotubes (TNTs) are cellular extensions enabling cytosol-to-cytosol intercellular interaction between numerous cell types including macrophages. Previous studies of hematopoietic stem and progenitor cell (HSPC) transplantation for the lysosomal storage disorder cystinosis have shown that HSPC-derived macrophages form TNTs to deliver cystinosin-bearing lysosomes to cystinotic cells, leading to tissue preservation. Here, we explored if macrophage polarization to either proinflammatory M1-like M(LPS/IFN?) or anti-inflammatory M2-like M(IL-4/IL-10) affected TNT-like protrusion formation, intercellular transport and, ultimately, the efficacy of cystinosis prevention. We designed new automated image processing algorithms used to demonstrate that LPS/IFN? polarization decreased bone marrow-derived macrophages (BMDMs) formation of protrusions, some of which displayed characteristics of TNTs, including cytoskeletal structure, 3D morphology and size. In contrast, co-culture of macrophages with cystinotic fibroblasts yielded more frequent and larger protrusions, as well as increased lysosomal and mitochondrial intercellular trafficking to the diseased fibroblasts. Unexpectedly, we observed normal protrusion formation and therapeutic efficacy following disruption of anti-inflammatory IL-4/IL-10 polarization in vivo by transplantation of HSPCs isolated from the Rac2-/- mouse model. Altogether, we developed unbiased image quantification systems that probe mechanistic aspects of TNT formation and function in vitro, while HSPC transplantation into cystinotic mice provides a complex in vivo disease model. While the differences between polarization cell culture and mouse models exemplify the oversimplicity of in vitro cytokine treatment, they simultaneously demonstrate the utility of our co-culture model which recapitulates the in vivo phenomenon of diseased cystinotic cells stimulating thicker TNT formation and intercellular trafficking from macrophages. Ultimately, we can use both approaches to expand the utility of TNT-like protrusions as a delivery system for regenerative medicine.

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Tunneling nanotube formation is driven by Eps8/IRSp53-dependent linear actin polymerization

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