Lee, Chan-Soo; Choi, Chang-Ki; Shin, Eun-Young; Schwartz, Martin Alexander; Kim, Eung-Gook (2021). CIL:26536, Mus musculus, fibroblast. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0ZW1JP2

To study the molecular mechanism by which nonmuscle myosin II (MII) regulates protrusion and adhesion dynamics in migrating cells, NIH3T3 cells were transfected with myc-tagged phosphomimetic constitutively active MLC mutants (MLCee, green). After 20' exposure to PDGF, which induces myosin-inactivation, stress fiber disassembly, and stimulates fibroblast motility, the cells were fixed and co-stained for betaPIX (a Rac1/Cdc42-specific GEF highly implicated in cell motility, red) and filamentous actin (blue). These findings help elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells. This image is original data file Fig. 7C "PDGF-induced dissociation of the MII–βPIX complex," in J. Cell Biol. 2010. Vol. 190(4):663–674

• 2021

• Choi, Chang-Ki
• Kim, Eung-Gook
• Lee, Chan-Soo
• Schwartz, Martin Alexander
• Shin, Eun-Young

Cells were cultured in DME (Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C in a humidified 5% CO2 incubator. For transfections, cells in 60-mm-diameter dishes or on fibronectin-coated coverslips were incu- bated with a mixture of DNA and LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. The cDNA for MLC was subcloned into pCMV-myc (Takara Bio Inc.). Mutant constructs of MLC were generated using a QuikChange site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. In this myc-tagged MLC mutant, Thr18/Ser19 were replaced by glutamic acids, and expressed in NIH3T3 cells.
In some experimental conditions, 16 h after replating onto fibronectin-coated coverslips, cells were treated with 50 ng/ml PDGF for 20 min. Cells were fixed 24–48 h after transfection using 3.7% paraformaldehyde in PBS for 15 min, permeabilized using 0.2% Triton X-100 in PBS for 2 min, blocked with 2% BSA in PBS, and co-stained for MLC (green), TRIO (red), and actin (blue). Images were captured by Zeiss LSM 710 confocal microscope with Plan-Apochromat 63X objective.

Preparation: formaldehyde fixed tissue; detergent permeabilized
Relation to intact cell: dispersed cells in vitro
Item type: recorded image
Imaging mode: confocal microscopy
Parameter imaged: fluorescence emission
Source of contrast: distribution of epitope; distribution of a specific protein
Visualization methods: Alexa Fluor 488; Alexa Fluor 546; phalloidin
Data qualification: Raw

• Cell Image Library Group ID: 8283

• Mus musculus

• Actin filament
• Fibroblast
• Lamellipodium
• Myosin II complex

• NIH/3T3
• Regulation of cell migration

• data
• image

• No linguistic content; Not applicable

Samplenumber: 26536

Doi: https://doi.org/10.6075/J0ZW1JP2

Source data

• Source Record in the Cell Image Library: https://doi.org/10.7295/W9CIL26536

Reference

• J. Cell Biol. 2010. Vol. 190(4):663–674 https://www.ncbi.nlm.nih.gov/pubmed/?term=0713598

Other resource

• BioStudies (previously JCB DataViewer): https://www.ebi.ac.uk/biostudies/studies/

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2022-08-12