The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

Abstract

Here we demonstrate that RNAi can be used in molecular studies of the giant single-celled ciliate Stentor coeruleus, revealing morphogenetic functions of Mob1 and highlighting the potential of this classical model for studies of morphogenesis and regeneration.

Lithium-induced developmental anomalies in the spirotrich ciliate Stylonychia lemnae (Ciliophora, Hypotrichida)

“…A further feature of universality is that many of the features of regulation and regeneration described by the polar coordinate model apply to organisms as diverse as insects and amphibia (Bryant et al 1981) and even to protozoa (Frankel et al 1984). Also, there are an increasing number of reports in which proteins (morphogens), such as EGF-like (epidermal growth factor) molecules (Wolpert 1989), receptors, Wnt proteins (Bajard et al 2014) and Mob1 (Mps one binder) (Slabodnick et al 2014) seem to be involved in patterning. Wnt signalling pathway has also been reported to play an important role in positional information in zebrafish somitogenesis (Bajard et al 2014).…”

Microfluidic guillotine for single-cell wound repair studies

“…We monitored the morphology of the cells manually under a bright-field stereomicroscope. Because the membrane of Stentor disintegrates quickly after death, we classify a cell to be alive and to have regenerated successfully if (i) it has restored membrane integrity demonstrated by beating cilia and (ii) it is either actively swimming or attached to a surface in its namesake trumpet-like shape 24 h after the cut, a typical duration of time for Stentor to complete regeneration (8). A grid was drawn on the bottom of the Petri dish to facilitate counting of live cells.…”

“…The survival rate is defined as N post−cut =2 · N pre−cut , where N pre−cut is the number of cells injected into the device, counted manually from the videos of the cutting process. As a benchmark, we compared cuts using our device to cuts performed by hand, the conventional wounding method in previous regeneration studies (8). To cut cells by hand, we heated a glass capillary over a flame and stretched it into a thin needle (tip diameter ∼30 μm), which was then cooled to room temperature before cutting through a cell that was immobilized in a 2% wt/vol methylcellulose solution.…”

“…Here, we use Stentor coeruleus as a model organism for single-cell wound repair studies because it satisfies such requirement (7)(8)(9). Stentor is a single-celled ciliate protozoan that is up to 1 mm long.…”

“…The genome of Stentor has recently been published (9). We have also demonstrated the utility of RNAi to knock down gene expression, by feeding Stentor bacteria containing an expression plasmid encoding dsRNA that targets genes of interest (7,8). Stentor thus offers a substantial technical advantage over Xenopus oocytes for high-throughput knockdown studies.…”