Fluorescent double labelling of F-actin in Foraminifera: evaluation of granular pattern F-actin organisation in reticulopodia

Abstract

Abstract. This paper presents novel results of experiments focused on actin cytoskeleton organisation in pseudopodial structures of foraminifera. The main aim of this research was to test the hypotheses proposed to explain the previously reported granular pattern of F-actin labelling in several species of foraminifera with SiR-actin fluorescent probe. These hypotheses include the possibility that SiR-actin enhances the F-actin polymerisation or that it binds to other than F-actin compounds within foraminiferal cells, resulting in capturing the staining artefacts. The series of replicated experiments conducted on small miliolid Quinqueloculina sp. included double staining of F-actin with SiR-actin and phalloidin-based probes, complemented with observations of reticulopodia under the polarised light to identify the granules showing birefringence. All of performed experiments demonstrate highly congruent results expressed in SiR-actin and phalloidin co-labelling patterns, especially pronounced with the small granular objects (SiR-actin labelled granules or ALGs). Furthermore, the birefringence strongly tends to characterise primarily areas stained with both of the probes. These results rule out staining artefacts and support existence of actin cytoskeleton associated with micrometre-size motile granules. We discuss possible implications of this granular pattern that facilitates efficient formation and fast reorganization of extended granuloreticulopodia and other pseudopodial structures. If this is the case, they are one of the key evolutionary adaptations of these organisms that most likely predates the evolution of the foraminiferal tests that started in the early Palaeozoic.