scholarly journals In vivo functional mapping of the conserved protein domains within murine Themis1

2014 ◽  
Vol 92 (8) ◽  
pp. 721-728 ◽  
Author(s):  
Ekaterina Zvezdova ◽  
Jan Lee ◽  
Dalal El‐Khoury ◽  
Valarie Barr ◽  
Itoro Akpan ◽  
...  
Keyword(s):  
Protein Domains ◽  
Functional Mapping

scholarly journals Functional Mapping of Adhesiveness on Live Cells Reveals How Guidance Phenotypes Can Emerge From Complex Spatiotemporal Integrin Regulation

2021 ◽  
Vol 9 ◽  
Author(s):  
Philippe Robert ◽  
Martine Biarnes-Pelicot ◽  
Nicolas Garcia-Seyda ◽  
Petra Hatoum ◽  
Dominique Touchard ◽  
...  
Keyword(s):  
Distance Effect ◽  
Cell Polarization ◽  
Functional Mapping ◽  
Live Cells ◽  
Long Distance ◽  
Adhesion Properties ◽  
Functional Assays ◽  
New Perspective

Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially in vivo. Here, we developed a novel in vitro method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/μm2 in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.

Functional mapping of glutamate receptors in dendrites of mouse neocortical neurons in vivo using two-photon microscope

2007 ◽  
Vol 58 ◽  
pp. S13
Author(s):  
Jun Noguchi ◽  
Takashi Miyazaki ◽  
Akira Nagaoka ◽  
Graham C.R. Ellis-Davies ◽  
Masanori Matsuzaki ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Functional Mapping ◽  
Two Photon ◽  
Neocortical Neurons

Carboxyl Tail Region of the Kv2.2 Subunit Mediates Novel Developmental Regulation of Channel Density

2004 ◽  
Vol 92 (6) ◽  
pp. 3446-3454 ◽  
Author(s):  
Judith T. Blaine ◽  
Alison D. Taylor ◽  
Angeles B. Ribera
Keyword(s):  
Molecular Mechanisms ◽  
Protein Domains ◽  
Functional Expression ◽  
Induced Voltage ◽  
Tail Region ◽  
Voltage Gated ◽  
Mature Neurons ◽  
Specific Regulation ◽  
Carboxyl Tail

Molecular mechanisms underlying the acquisition of stable electrical phenotypes in developing neurons remain poorly defined. As Xenopus embryonic spinal neurons mature, they initially exhibit dramatic changes in excitability due to a threefold increase in voltage-gated potassium current ( IKv) density. Later when mature neurons begin synapse formation, IKv density remains stable. Elevation of Kv1.1 and Kv2.1 RNA levels indicates that excess transcript levels of these Kv genes can increase current density in both young and mature neurons. In contrast, Kv2.2 overexpression increases IKv density in young but not mature neurons despite the presence of protein translated from injected RNA at this stage. Because protein domains can determine biophysical as well as subcellular localization properties of channel subunits, we tested whether a region of the Kv2.2 subunit regulated functional expression in mature neurons. We focused on the large cytoplasmic carboxy tail, a region that differs most between Kv2.2 and the structurally related Kv2.1 subunit. Chimeric Kv2 subunits were generated in which different regions of the large cytoplasmic carboxyl tail were exchanged between Kv2.1 and Kv2.2 subunits. All chimeric Kv2 subunits induced voltage-gated potassium currents when expressed heterologously in oocytes. In vivo chimeric subunits increased IKv density in young neurons on overexpression in the developing embryo. In contrast, in mature neurons, only those chimeras lacking a domain in the proximal carboxy terminus, proxC, increased IKv density when overexpressed. Thus the proxC domain mediates developmental and subunit-specific regulation of IKv and identifies a novel function for protein domains.

scholarly journals Functional mapping of the trichromatic cone mosaic in vivo

2014 ◽  
Vol 14 (15) ◽  
pp. 21-21 ◽  
Author(s):  
W. Tuten ◽  
W. Harmening ◽  
R. Sabesan ◽  
L. Sincich ◽  
A. Roorda
Keyword(s):  
Functional Mapping ◽  
Cone Mosaic
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