Extracellular signal-regulated kinase (ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under ...steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution (SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue.
The progress in imaging technology with fluorescent proteins has uncovered a wide range of biological processes in developmental biology. In particular, genetically‐encoded biosensors based on the ...principle of fluorescence resonance energy transfer (FRET) have been used to visualize spatial and temporal dynamics of intracellular signaling in living cells. However, development of sensitive FRET biosensors and their application to developmental biology remain challenging tasks, which has prevented their widespread use in developmental biology. In this review, we first overview general procedures and tips of imaging with FRET biosensors. We then describe recent advances in FRET imaging – namely, the use of optimized backbones for intramolecular FRET biosensors and transposon‐mediated gene transfer to generate stable cell lines and transgenic mice expressing FRET biosensors. Finally, we discuss future perspectives of FRET imaging in developmental biology.
Genetically-encoded biosensors based on the principle of Förster resonance energy transfer (FRET) have been widely used in biology to visualize the spatiotemporal dynamics of signaling molecules. ...Despite the increasing multitude of these biosensors, their application has been mostly limited to cultured cells with transient biosensor expression, due to particular difficulties in the development of transgenic mice that express FRET biosensors. In this study, we report the efficient generation of transgenic mouse lines expressing heritable and functional biosensors for ERK and PKA. These transgenic mice were created by the cytoplasmic co-injection of Tol2 transposase mRNA and a circular plasmid harbouring Tol2 recombination sites. High expression of the biosensors in a wide range of cell types allowed us to screen newborn mice simply by inspection. Observation of these transgenic mice by two-photon excitation microscopy yielded real-time activity maps of ERK and PKA in various tissues, with greatly improved signal-to-background ratios. Our transgenic mice may be bred into diverse genetic backgrounds; moreover, the protocol we have developed paves the way for the generation of transgenic mice that express other FRET biosensors, with important applications in the characterization of physiological and pathological signal transduction events in addition to drug development and screening.
The constituents of the oncogene signal transduction pathway are promising targets for anticancer drugs. Despite the wealth of available knowledge regarding their molecular properties, the ...spatiotemporal regulation of the signaling molecules remains elusive. Biosensors based on the principle of FRET have been developed to visualize the activities of the signaling molecules in living cells. However, difficulties in the development of sensitive FRET biosensors have prevented their widespread use in cancer research. The lack of cell lines constitutively expressing a FRET biosensor has also limited their use. In this review, we will introduce the principle of FRET‐based biosensors, describe an optimized backbone of the FRET biosensors, techniques to express FRET biosensors stably in the cells, and discuss the future perspectives of FRET biosensors in cancer research. (Cancer Sci 2012; 103: 614–619)
Bidirectional control of integrin activation plays crucial roles in cell adhesive behaviors, but how integrins are specifically regulated by inside-out and outside-in signaling has not been fully ...understood. Here, we report distinct bidirectional regulation of major lymphocyte homing receptors LFA1 and α4β7 in primary T cells. A small increase of Rap1 activation in L-selectin-mediated tether/rolling was boosted by the outside-in signaling from ICAM1-interacting LFA1 through subsecond, simultaneous activation of Rap1 GTPase and talin1, but not kindlin-3, resulting in increased capture and slowing. In contrast, none of them were required for tether/rolling by α4β7 on MAdCAM1. High Rap1 activation with chemokines or the loss of Rap1-inactivating proteins Rasa3 and Sipa1 increased talin1/kindlin-3-dependent arrest with high-affinity binding of LFA1 to membrane-anchored ICAM1. However, despite increased affinity of α4β7, activated Rap1 severely suppressed adhesion on MAdCAM1 under shear flow, indicating the critical importance of a sequential outside-in/inside-out signaling for α4β7.
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•Kindlin-3 is partially involved in attachment to high-density ICAM1 and HEV•Co-occupancy of LFA1 with ICAM1 and talin1 activates Rap1 and induces slow rolling•Distinct Rap1 impacts on affinity of integrins with different outcomes under flow
Kamioka et al. reveal that Rap1 and integrin adaptors talin1 and kindlin-3 differently regulate two major homing integrins, LFA1 and α4β7, in T cells, resulting in distinct impacts by Rap1 on affinity of LFA1 and α4β7, generating promotion or inhibition of adhesion of T cells under flow conditions.
Significance Selection of actions that allow the seeking of rewards and avoidance of uncomfortable environments is a fundamental animal behavior. Here, we report an in vivo method, in which the ...activities of PKA and ERK were optically recorded by microendoscopy of Förster resonance energy transfer responses of biosensors in distinct D1 and D2 dopamine receptor-expressing neurons of the dorsal striatum. The PKA and ERK were coordinately but reciprocally regulated not only by rewarding and aversive stimuli but also between the two parallel projection neurons. Importantly, the cell type-specific regulation of PKA and ERK was causally linked to active and indifferent mating reactions of male mice. The dynamic modulation of PKA and ERK in the striatum underlies the selection of alternative actions.
The selection of reward-seeking and aversive behaviors is controlled by two distinct D1 and D2 receptor-expressing striatal medium spiny neurons, namely the direct pathway MSNs (dMSNs) and the indirect pathway MSNs (iMSNs), but the dynamic modulation of signaling cascades of dMSNs and iMSNs in behaving animals remains largely elusive. We developed an in vivo methodology to monitor Föörster resonance energy transfer (FRET) of the activities of PKA and ERK in either dMSNs or iMSNs by microendoscopy in freely moving mice. PKA and ERK were coordinately but oppositely regulated between dMSNs and iMSNs by rewarding cocaine administration and aversive electric shocks. Notably, the activities of PKA and ERK rapidly shifted when male mice became active or indifferent toward female mice during mating behavior. Importantly, manipulation of PKA cascades by the Designer Receptor recapitulated active and indifferent mating behaviors, indicating a causal linkage of a dynamic activity shift of PKA and ERK between dMSNs and iMSNs in action selection.
Rap1-GTPase activates integrins and plays an indispensable role in lymphocyte trafficking, but the importance of Rap1 inactivation in this process remains unknown. Here we identified the ...Rap1-inactivating proteins Rasa3 and Sipa1 as critical regulators of lymphocyte trafficking. The loss of Rasa3 and Sipa1 in T cells induced spontaneous Rap1 activation and adhesion. As a consequence, T cells deficient in Rasa3 and Sipa1 were trapped in the lung due to firm attachment to capillary beds, while administration of LFA1 antibodies or loss of talin1 or Rap1 rescued lung sequestration. Unexpectedly, mutant T cells exhibited normal extravasation into lymph nodes, fast interstitial migration, even greater chemotactic responses to chemokines and sphingosine-1-phosphate, and entrance into lymphatic sinuses but severely delayed exit: mutant T cells retained high motility in lymphatic sinuses and frequently returned to the lymph node parenchyma, resulting in defective egress. These results reveal the critical trafficking processes that require Rap1 inactivation.
Two-photon excitation microscopy was used to visualized two different modes of invasion at perivascular and intraparenchymal regions of rat C6 glioblastoma cells that were orthotopically implanted ...into rat brains. Probes based on the principle of Förster resonance energy transfer (FRET) further revealed that glioblastoma cells penetrating the brain parenchyma showed higher Rac1 and Cdc42 activities and lower RhoA activity than those advancing in the perivascular regions. This spatial regulation of Rho-family GTPase activities was recapitulated in three-dimensional spheroid invasion assays with rat and human glioblastoma cells, in which multipod glioblastoma cells that invaded the gels and led the other glioblastoma cells exhibited higher Rac1 and Cdc42 activities than the trailing glioblastoma cells. We also studied the Cdc42-specific guanine nucleotide exchange factor Zizimin1 (also known as DOCK9) as a possible contributor to this spatially controlled activation of Rho-family GTPases, because it is known to play an essential role in the extension of neurites. We found that shRNA-mediated knockdown of Zizimin1 inhibited formation of pseudopodia and concomitant invasion of glioblastoma cells both under a 3D culture condition and in vivo. Our results suggest that the difference in the activity balance of Rac1 and Cdc42 versus RhoA determines the mode of glioblastoma invasion and that Zizimin1 contributes to the invasiveness of glioblastoma cells with high Rac1 and Cdc42 activities.
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