Images & Movies > Live cell imaging

Confocal, deconvolution, and wide-field microscopy of living cell cultures

Live cell imaging of actin dynamics during purse-string wound closure in EGFP-ß-actin-expressing Caco-2 cells. Within 2 minutes after wounding, actin polymerization occurred at the wound edge. By 8 minutes, a continuous ring of actin was present and the wound rounded, indicating the development of circumferential tension. This was followed by contraction of the actomyosin ring as the wound closed. Cells surrounding the wound also contracted. Most activity occurred within the first 30 minutes, and wound closure was complete by 60 minutes. The wound-healing process was associated with stretching and flattening of some cells, resulting in a shift out of the plane of focus at the last time points. (<a target="_blank" href="http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/Gastro05.pdf?ver=2015-09-10-182343-387">Gastroenterology 2005;128:987-1001.</a>)

In vitro purse-string wound closure

Live cell imaging of actin dynamics during purse-string wound closure in EGFP-ß-actin-expressing Caco-2 cells treated with the ROCK inhibitor Y27632. Within minutes of wounding, foci of actin polymerization developed in regions along the wound edge, similar to that seen in wounded monolayers that were not treated with Y27632. However, these site were unstable, a complete actomyosin ring did not form, and cells surrounding the wound did not exhibit any meaningful contraction. Wound edges did not round, and no significant wound closure occurred. Focal filopodial extensions formed, but were transient and did not contribute to wound closure. (<a target="_blank" href="http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/Gastro05.pdf?ver=2015-09-10-182343-387">Gastroenterology 2005;128:987-1001.</a>)

Rho kinase is required for wound closure

Live cell imaging of actin dynamics during purse-string wound closure in EGFP-ß-actin-expressing Caco-2 cells treated with the myosin light chain kinase inhibitor PIK. Actin ring assembly and early wound edge rounding proceeded normally. However, at later times the ring fragmented and the wound enlarged, appearing to spring back to the original size. Compare to wounded monolayers that were not treated with PIK. (<a target="_blank" href="http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/Gastro05.pdf?ver=2015-09-10-182343-387">Gastroenterology 2005;128:987-1001.</a>)

Myosin light chain kinase drives wound closure

Live imaging of Caco-2 cells expressing GFP-ZO-1. A small portion of the tight junction is photobleached at the start of the movie. Fluorescence recovery after photobleaching (FRAP)occurs rapidly, with ~60% recovery in 10 minutes. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/PNAS2010.pdf?ver=2015-09-10-182343-323">Proc Natl Acad Sci U S A 2010;107:8237-41.</a>)

ZO-1 FRAP (in vitro)

Live imaging of MDCK monolayers stably expressing EGFP-ß-actin (green) and mRFP1-occcludin (red). Simultaneous measurements show that transepithelial electrical resistance is stable for at least 45 minutes during this imaging. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

Live imaging of occludin and actin

Live imaging of MDCK monolayers stably expressing EGFP-ß-actin (green) and mRFP1-occcludin (red) collected while simultaneously measuring transepithelial electrical resistance. Addition of latrunculin A (latA), to inhibit ß-actin polymerization, causes transepithelial electrical resistance to fall within less than 5 min. This is associated with occludin endocytosis, but no qualitative change in F-actin distribution. The shape changes that occur at the end of the movie occur long after loss of transepithelial electrical resistance and are best interpreted as either secondary events or effects unrelated to tight junctions regulation. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

LatA causes occludin endocytosis

Live imaging of a portion of a MDCK cell expressing mRFP1-occcludin. Latrunculin A (latA) treatment induces occludin endocytosis, shown at high magnification, within less than 5 min. The movie covers an interval of 1.5 minutes beginning 5 minutes after latrunculin A addition. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

LatA causes occludin endocytosis

Live imaging of MDCK monolayers stably expressing EGFP-ß-actin (green) and mRFP1-ZO-1 (red) collected while simultaneously measuring transepithelial electrical resistance. Addition of latrunculin A (latA), to inhibit ß-actin polymerization, causes transepithelial electrical resistance to fall within less than 5 min. This is not associated with any qualitative change in F-actin or ZO-1 distribution. The shape changes that occur at the end of the movie occur long after loss of transepithelial electrical resistance and are best interpreted as either secondary events or effects unrelated to tight junctions regulation. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

LatA does not acutely disrupt ZO-1 distribution

Live imaging of MDCK monolayers stably expressing EGFP-claudin-1 (green) and mRFP1-occcludin (red) collected while simultaneously measuring transepithelial electrical resistance. Addition of latrunculin A (latA), to inhibit ß-actin polymerization, causes transepithelial electrical resistance to fall within less than 5 min. This is associated with occludin endocytosis, but no qualitative change in claudin-1 distribution. The shape changes that occur at the end of the movie occur long after loss of transepithelial electrical resistance and are best interpreted as either secondary events or effects unrelated to tight junctions regulation. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

LatA does not disrupt claudin-1 distribution

Live imaging of MDCK monolayers stably expressing EGFP-ß-actin (green) and mRFP1-occcludin (red) held at 4°C while simultaneously measuring transepithelial electrical resistance. Addition of latrunculin A (latA), to inhibit ß-actin polymerization, does not causes transepithelial electrical resistance to fall at 4°C. Occludin endocytosis and late shape changes are also blocked at 4°C. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

Occludin internalization is blocked at 4°C

Live imaging of MDCK monolayers stably expressing caveolin-1-EGFP (green) and mRFP1-ZO-1 (red). Addition of latrunculin A, to inhibit ß-actin polymerization, causes co-internalization of caveolin-1 and occludin. The movie covers an interval of 3.5 minutes beginning approximately 5 minutes after latrunculin A addition. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

Occludin is co-internalized with caveolin-1

Live imaging of MDCK monolayers stably expressing caveolin-1-EGFP (green) and mRFP1-ZO-1 (red). Addition of latrunculin A, to inhibit ß-actin polymerization, causes co-internalization of caveolin-1 and occludin. As shown in this move, occludin is then removed from the caveolin-1-containing vesicles, suggesting that it is trafficked to a separate intracellular compartment. The movie covers an interval of 2.5 minutes beginning approximately 15 minutes after latrunculin A addition. (<a target="_blank" href=" http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/MBC05.pdf?ver=2015-09-10-182342-513">Mol Biol Cell 2005;16:3919-36.</a>)

Occludin traffics through cav-1 vesicles

In vitro live cell imaging showing GFP-tagged gammadelta T cells (green) migrating within a Caco-2 monolayer (phase microscopic image), viewed from the apical surface. A gammadelta T cell moving in and out of the epithelial compartment is marked by a white asterisk when within the epithelial monolayer. (<a target="_blank" href="http://www.jrturnerlab.com/Portals/0/Uploads/documents/pubs-pdfs/PNAS2012.pdf?ver=2015-09-10-182343-403">Proc Natl Acad Sci U S A 2012;109:7097-102.</a>)

Intraepithelial T cell migration in vitro