Authors

Harada, Y., Tanaka, Y., Terasawa, M., Pieczyk, M., Habiro, K., Katakai, T., … Fukui, Y.

Affiliations

Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan; Research Centre for Advanced Immunology, Kyushu University, Fukuoka, Japan; Theodor Kocher Institute, University of Bern, Bern, Switzerland; Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, Osaka, Japan; RIKEN Systems and Structural Biology Centre, Yokohama, Japan; and Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Application Area

Neuroscience

FRET

Abstract

To migrate efficiently through the interstitium, dendritic cells (DCs) constantly adapt their shape to the given structure of the extracellular matrix and follow the path of least resistance. It is known that this amoeboid migration of DCs requires Cdc42, yet the upstream regulators critical for localization and activation of Cdc42 remain to be determined. Mutations of DOCK8, a member of the atypical guanine nucleotide exchange factor family, causes combined immunodeficiency in humans. In the present study, we show that DOCK8 is a Cdc42-specific guanine nucleotide exchange factor that is critical for interstitial DC migration. By generating the knockout mice, we found that in the absence of DOCK8, DCs failed to accumulate in the lymph node parenchyma for T-cell priming. Although DOCK8-deficient DCs migrated normally on 2-dimensional surfaces, DOCK8 was required for DCs to crawl within 3-dimensional fibrillar networks and to transmigrate through the subcapsular sinus floor. This function of DOCK8 depended on the DHR-2 domain mediating Cdc42 activation. DOCK8 deficiency did not affect global Cdc42 activity. However, Cdc42 activation at the leading edge membrane was impaired in DOCK8-deficient DCs, resulting in a severe defect in amoeboid polarization and migration. Therefore, DOCK8 regulates interstitial DC migration by controlling Cdc42 activity spatially.

Extract

… “used for CFP and YFP. Cells were illuminated at 440 nm excitation with a CoolLED. After recordings were made, ratio images of FRET/CFP were created with MetaMorph software and were used to represent the efficiency of the FRET”….

Product Associated Features

pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.

Diascopic Technique

-

Live Cell Issues

-

Product Type

pE-100

Journal

Blood

Year of Publication

2012

Country of Publication

Japan