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. 2016;136:143-59.
doi: 10.1016/bs.mcb.2016.03.021. Epub 2016 Jun 14.

Live Cell Imaging of Septin Dynamics in Ustilago Maydis

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Live Cell Imaging of Septin Dynamics in Ustilago Maydis

S Baumann et al. Methods Cell Biol. .

Abstract

Septins are highly conserved cytoskeletal proteins involved in a variety of biological processes such as cell polarization and cytokinesis. In humans, functional defects in these proteins have been linked to cancer and neuronal diseases. In recent years, substantial progress has been made in studying the structure of septin subunits and the formation of defined heteromeric building blocks. These are assembled into higher-order structures at distinct subcellular sites. An important microscopic approach in studying septin assembly and dynamics is the use of septins tagged with fluorescent proteins. This revealed, eg, that septins form rings during cytokinesis and that septins build extended filaments partially colocalizing with actin cables and microtubules. Here, we describe extensive live cell imaging of septins in the model microorganism Ustilago maydis. We present techniques to study dynamic localization of protein and septin mRNA on shuttling endosomes as well as colocalization of proteins at these highly motile units. Moreover, FLIM-FRET experiments for analyzing local protein interactions are presented. Importantly, these imaging approaches transfer well to other fungal and animal model systems for in vivo analysis of septin dynamics.

Keywords: Basidiomycete; Dual color microscopy; Endosomes; FLIM-FRET; Microtubules; RNA live imaging; Rab5; Real-time movies; Septin dynamics; mRNA transport.

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