RNA localization in conﬁned cells depends on cellular mechanical activity and contributes to conﬁned migration

SUMMARY Cancer cells experience mechanical conﬁning forces during metastasis and, consequently, can alter their migratory mechanisms. Localization of numerous mRNAs to cell protrusions contributes to cell polarization and migration and is controlled by proteins that can bind RNA and/or cytoskeletal elements, such as the adenomatous polyposis coli (APC). Here, we demonstrate that peripheral localization of APC-dependent RNAs in cells within conﬁned microchannels is cell type dependent. This varying phenotype is determined by the levels of a detyrosinated tubulin network. We show that this network is regulated by mechanoactivity and that cells with mechanosensitive ion channels and increased myosin II activity direct peripheral localization of the RAB13 APC-dependent RNA. Through speciﬁc mislocalization of the RAB13 RNA, we show that peripheral RNA localization contributes to conﬁned cell migration. Our results indicate that a cell’s mechanical activity determines its ability to peripherally target RNAs and utilize them for movement in conﬁnement. protrusions contractile, In this study, we sought to understand how RNA localization is controlled by the mechanical cue of conﬁnement. We employed microﬂuidic microchannel devices to model unconﬁned and conﬁned environments. We imaged RNAs in two cells lines: (1) the MDA-MB-231 metastatic breast cancer cell line, which can use microtubule-based methods 2012) and/or an osmotic engine model 2014) to migrate in conﬁned spaces, and (2) the A375 metastatic melanoma line, which utilizes ameboid-based myosin II contractility in conﬁnement We show that RNA localization patterns change in conﬁnement in a cell type-dependent manner. We focused on the APC-dependent RAB13 RNA, which remains peripherally localized only in conﬁned A375 cells. We show that RAB13 RNA localization depends on detyrosinated microtubules and the mechanical state of cells. Speciﬁcally, the Piezo1 mechanosensitive ion channel and myosin II activity act redundantly to upregulate the detyrosinated tubulin network in conﬁnement, leading to peripheral RAB13 RNA localization. This mechanism functionally contributes to conﬁned migration of A375 cells. By contrast, conﬁned MDA-MB-231 cells do not exhibit peripheral RAB13 RNA and do not rely on it for migration in narrow spaces. This work highlights the contribution of peripherally localized RNAs during conﬁned migration and indicates that silicon wafer. A MA-4 SUSS aligner was used to UV crosslink the SU-8 through the microchannel mask. Uncrosslinked photoresist was washed away with SU-8 developer (MicroChem Corp). Channel widths of 50 or 3 m m by 200 m m in length were conﬁrmed using a proﬁlometer. A second 50 m m layer of SU-8 3025 negative photoresist (MicroChem Corp) was spun onto the wafer and the mask containing the feed lines was aligned to the microchannel features and exposed to UV light. Any remaining uncrosslinked photoresist was again washed away with SU-8 developer. Wafers were silanized overnight with tridecaﬂuoro-1,1,2,2,tetrahydrooctyl-1-trichlorosi-lane (Sigma) in a vacuum desiccator. Polydimethylsiloxane (Sylgard (cid:3) 184, Ellsworth Adhesives) was mixed at a 10:1 base to crosslinker ratio, poured over the silicon wafer, placed into a vacuum desiccator for 1 hour and then baked at 80 (cid:2) C for at least 2 hours. PDMS was removed from the wafer, cleaned with 100% ethanol and water, dried at 80 (cid:2) C for 5 minutes, and then plasma treated for 2.5 minutes. The PDMS devices were then bound to glass coverslips for 5 minutes and the PDMS coverslip unit was UV sterilized for 10 minutes. standard error (SEM) and data represents three individual trials that have been pooled. Individual replicates generally show consistent trends, even though they might not independently reach statistical signiﬁcance. Prism 9 (GraphPad) was used for all statistical analysis; the signiﬁcance level was set at 0.05. For multiple comparisons, a two-way ANOVA with (cid:1) Sı´da´k’s test was performed. For single comparisons, an unpaired t-test assuming equal standard deviations was used.