Figure 9.
Membrane-encapsulated mtDNA promotes increased trafficking and invasiveness in a TLR9-dependent mechanism. (a) Protocol for encapsulation of mtDNA within POPC liposomes, followed by removal of surface-associated mtDNA by treatment with DNase immobilized to Affi-Gel 10 agarose beads. (b) POPC liposomes were formed in the absence and presence of mtDNA and treated with immobilized DNase as indicated. The mtDNA content of these was then determined using qPCR with primers recognizing the mitochondrial ND1 gene. Values represent mean ± SEM. (c) Glutamine-starved cells were incubated overnight with DNase-treated liposomes (lipos) that had previously been formed in the absence or presence (+mtDNA) of mtDNA. Recycling of MT1-MMP, α5β1 integrin, and active α5β1 integrin was determined using cell surface biotinylation/capture ELISA approaches. The proportion of receptors recycled to the plasma membrane is expressed as a percentage of the pool of the receptor labeled during the internalization period. Values are mean ± SEM; n = 4 independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; unpaired t test. (d) Glutamine-starved cells were incubated for 3 d with DNase-treated liposomes (lipos) formed in the absence or presence of mtDNA. ODN-TTAGGG-A151 (TLR9 antagonist; 1 µM) or ODN-TTAGGG-control (Ctrl antagonist; 1 µM) was also included as indicated. Cells were then transfected with mCherry-MT1-MMP and imaged using TIRF microscopy as in Fig. 6 b. Values represent mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; two-way ANOVA with Dunn’s multiple comparison test. (e) Glutamine-starved cells were treated with mtDNA-containing liposomes in the presence and absence of TLR9 antagonist as in d and transfected with siRNAs targeting TLR9 (siTLR9) or nontargeting siRNA (siNT). Cells were then plated into fibroblast-derived ECM, and the extension of invasive protrusions at the cell front was determined as in Fig. 6 d. Representative stills are presented. Bar, 30 µm. Whiskers are the 10th–90th percentiles; + represents the mean; n = 3. ****, P < 0.0001; two-way ANOVA with Dunn’s multiple comparison test.

Membrane-encapsulated mtDNA promotes increased trafficking and invasiveness in a TLR9-dependent mechanism. (a) Protocol for encapsulation of mtDNA within POPC liposomes, followed by removal of surface-associated mtDNA by treatment with DNase immobilized to Affi-Gel 10 agarose beads. (b) POPC liposomes were formed in the absence and presence of mtDNA and treated with immobilized DNase as indicated. The mtDNA content of these was then determined using qPCR with primers recognizing the mitochondrial ND1 gene. Values represent mean ± SEM. (c) Glutamine-starved cells were incubated overnight with DNase-treated liposomes (lipos) that had previously been formed in the absence or presence (+mtDNA) of mtDNA. Recycling of MT1-MMP, α5β1 integrin, and active α5β1 integrin was determined using cell surface biotinylation/capture ELISA approaches. The proportion of receptors recycled to the plasma membrane is expressed as a percentage of the pool of the receptor labeled during the internalization period. Values are mean ± SEM; n = 4 independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; unpaired t test. (d) Glutamine-starved cells were incubated for 3 d with DNase-treated liposomes (lipos) formed in the absence or presence of mtDNA. ODN-TTAGGG-A151 (TLR9 antagonist; 1 µM) or ODN-TTAGGG-control (Ctrl antagonist; 1 µM) was also included as indicated. Cells were then transfected with mCherry-MT1-MMP and imaged using TIRF microscopy as in Fig. 6 b. Values represent mean ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; two-way ANOVA with Dunn’s multiple comparison test. (e) Glutamine-starved cells were treated with mtDNA-containing liposomes in the presence and absence of TLR9 antagonist as in d and transfected with siRNAs targeting TLR9 (siTLR9) or nontargeting siRNA (siNT). Cells were then plated into fibroblast-derived ECM, and the extension of invasive protrusions at the cell front was determined as in Fig. 6 d. Representative stills are presented. Bar, 30 µm. Whiskers are the 10th–90th percentiles; + represents the mean; n = 3. ****, P < 0.0001; two-way ANOVA with Dunn’s multiple comparison test.

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