The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

Niño, Jorge Luis Galeano; Tay, Szun S.; Tearle, Jacqueline L.E.; Xie, Jianling; Govendir, Matt A.; Kempe, Daryan; Mazalo, Jessica K; Drew, Alexander P.; Colakoglu, Feyza; Kummerfeld, Sarah; Proud, Christopher G.; Biro, Maté

doi:10.1242/jcs.238014

Cited by 10 publications

(5 citation statements)

References 57 publications

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“…The current study provides the potential groundwork to continue studying the relevance of DAT in immunity. Future studies could employ coculture systems and in vivo tools such as fluorescent reporters for T cell activation ( 80 ) to unravel further roles for the various modes of DAT activity on macrophages.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of the biogenic amine transporters on human macrophages

Mackie¹,

Gopinath²,

Montas³

et al. 2022

JCI Insight

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Monocyte-derived macrophages are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remains nebulous. In the central nervous system (CNS), biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine (NET) and dopamine (DAT) transporters on human monocyte-derived macrophages. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured monocyte-derived macrophages (MDMs), but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immuno-modulatory mechanism in response to lipopolysaccharide (LPS). LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the pro-inflammatory response to LPS. Collectively, our data introduce a potential role for DAT in the regulation of innate immunity.

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Section: Discussionmentioning

confidence: 99%

Functional characterization of the biogenic amine transporters on human macrophages

Mackie¹,

Gopinath²,

Montas³

et al. 2022

JCI Insight

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“…LLSM experiments were either performed on a custom-built system described in [40], or on a Zeiss Lattice Light Sheet 7 microscope (Zeiss, Oberkochen, Germany). OT1-Lifeact-GFP T cells [41] labelled with CellTracker Deep Red dye were excited at 642 nm, OT1-mT/mG at 561 nm and plain OT1-Lifeact-GFP T cells at 488 nm. For all experiments performed on the home-built system, Point Spread Functions were measured using 200 nm Tetraspeck beads.…”

Section: Discussionmentioning

confidence: 99%

T cell morphodynamics reveal periodic shape oscillations in 3D migration

Cavanagh¹,

Kempe²,

Mazalo³

et al. 2022

Preprint

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T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralise infected and cancerous cells. However, there is limited quantitative understanding of the migration process in 3D extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the 3D morphodynamics of migrating T cells at high spatiotemporal resolution. We first developed a new shape descriptor based on spherical harmonics, incorporating key polarisation information of the uropod. We found that the shape space of T cells is low-dimensional. At the behavioural level, run-and-stop migration modes emerge at ∼150 s, and we mapped the morphodynamic composition of each mode using multiscale wavelet analysis, finding 'stereotyped' motifs. Focusing on the run mode, we found morphodynamics oscillating periodically (every ∼100 s) that can be broken down into a biphasic process: front-widening with retraction of the uropod, followed by a rearward surface motion and forward extension, where intercalation with the ECM in both of these steps likely facilitates forward motion. Further application of these methods may enable the comparison of T cell migration across different conditions (e.g. differentiation, activation, tissues, and drug treatments), and improve the precision of immunotherapeutic development.

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“…LLSM experiments were either performed on a custom-built system described in [ 40 ], or on a Zeiss Lattice Light Sheet 7 microscope (Zeiss, Oberkochen, Germany). OT1-Lifeact-GFP T cells [ 41 ] labelled with CellTracker Deep Red dye were excited at 642 nm, OT1-mT/mG at 561 nm and plain OT1-Lifeact-GFP T cells at 488 nm. For all experiments performed on the home-built system, Point Spread Functions (PSFs) were measured using 200 nm Tetraspeck beads.…”

Section: Methodsmentioning

confidence: 99%

“…Primary murine OT1-Lifeact-GFP and OT1-mT/mG cytotoxic T cells were isolated and cultured as previously described [ 41 ]. All imaging was done with T cells cultured over 6 or 7 days.…”

Section: Methodsmentioning

confidence: 99%

T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration

Cavanagh

Kempe

Mazalo

et al. 2022

J. R. Soc. Interface.

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T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralize infected and cancerous cells. However, there is limited quantitative understanding of the migration process in three-dimensional extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the three-dimensional morphodynamics of migrating T cells at high spatio-temporal resolution. We first developed a new shape descriptor based on spherical harmonics, incorporating key polarization information of the uropod. We found that the shape space of T cells is low-dimensional. At the behavioural level, run-and-stop migration modes emerge at approximately 150 s, and we mapped the morphodynamic composition of each mode using multiscale wavelet analysis, finding ‘stereotyped’ motifs. Focusing on the run mode, we found morphodynamics oscillating periodically (every approx. 100 s) that can be broken down into a biphasic process: front-widening with retraction of the uropod, followed by a rearward surface motion and forward extension, where intercalation with the ECM in both of these steps likely facilitates forward motion. Further application of these methods may enable the comparison of T cell migration across different conditions (e.g. differentiation, activation, tissues and drug treatments) and improve the precision of immunotherapeutic development.

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The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation

Cited by 10 publications

References 57 publications

Functional characterization of the biogenic amine transporters on human macrophages

Functional characterization of the biogenic amine transporters on human macrophages

T cell morphodynamics reveal periodic shape oscillations in 3D migration

T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration

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