Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naïve T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients ('sparkles') as cells migrate.
- Joseph L Dynes
- Michael D Cahalan
- Michael D Cahalan
- Michael D Cahalan
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Use of blood samples from healthy human subjects has been approved by the University of California, Irvine Institutional Review Board (UCI IRB HS #1995-459). All animal procedures were approved by the UCI Institutional Animal Care and Use committee (IACUC) (protocol #1998-1366-11).
Human subjects: Human blood was prepared using support from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NIH Grant UL1 TR000153).
- Michael L Dustin, University of Oxford, United Kingdom
© 2017, Dong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.
Different organelles traveling through neurons exhibit distinct properties in vitro, but this has not been investigated in the intact mammalian brain. We established simultaneous dual color two-photon microscopy to visualize the trafficking of Neuropeptide Y (NPY)-, LAMP1-, and RAB7-tagged organelles in thalamocortical axons imaged in mouse cortex in vivo. This revealed that LAMP1- and RAB7-tagged organelles move significantly faster than NPY-tagged organelles in both anterograde and retrograde direction. NPY traveled more selectively in anterograde direction than LAMP1 and RAB7. By using a synapse marker and a calcium sensor, we further investigated the transport dynamics of NPY-tagged organelles. We found that these organelles slow down and pause at synapses. In contrast to previous in vitro studies, a significant increase of transport speed was observed after spontaneous activity and elevated calcium levels in vivo as well as electrically stimulated activity in acute brain slices. Together, we show a remarkable diversity in speeds and properties of three axonal organelle marker in vivo that differ from properties previously observed in vitro.