Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells

ABSTRACT This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.

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Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear

Nature Communications ◽

10.1038/ncomms7873 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 27

Author(s):

Meghan C. Drummond ◽

Melanie Barzik ◽

Jonathan E. Bird ◽

Duan-Sun Zhang ◽

Claude P. Lechene ◽

...

Keyword(s):

Inner Ear ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Actin Dynamics ◽

Length Regulation

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Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Journal of Visualized Experiments ◽

10.3791/1325 ◽

2009 ◽

Cited By ~ 3

Author(s):

James Lim ◽

Gaudenz Danuser

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Actin Dynamics

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Actin chromobody imaging reveals sub-organellar actin dynamics

10.1101/639278 ◽

2019 ◽

Cited By ~ 2

Author(s):

Cara R. Schiavon ◽

Tong Zhang ◽

Bing Zhao ◽

Leonardo Andrade ◽

Melissa Wu ◽

...

Keyword(s):

Cell Migration ◽

Fluorescence Microscopy ◽

Actin Cytoskeleton ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Live Cell ◽

Actin Dynamics ◽

Spatiotemporal Resolution ◽

In Cells

AbstractThe actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi.

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