The actin cytoskeleton organization and disorganization properties of the photosynthetic dinoflagellateSymbiodinium kawagutiiin culture

Abstract

The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze-fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)-phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells.

Transient transformation of cultured photosynthetic dinoflagellates (Symbiodinium spp.) with plant-targeted vectors

“…Further evidence of heterologous expression was the fact that the transformed cells observed under epifluorescence revealed a reticular grid-type pattern of heavily bundled microfilaments. The organization patterns were different from those observed with the GFP-AtRACK1C expression and very similar to what has been observed with FITC-phalloidin and anti-actin antibodies in S. kawagutii cells (Villanueva et al 2014). Taken together, our data demonstrate that foreign gene introduction to different Symbiodinium clades with two distinct expression vectors harboring different resistance genes, mediated by vigorous shaking with glass beads and PEG, provide a successful and reproducible procedure for transiently transforming Symbiodinium spp.…”

Contribution of the cytoskeleton to mechanosensitivity reported by dinoflagellate bioluminescence

“…F‐actin has been imaged and quantified in cells with well‐developed actin filaments and structures (Connacher, Tay, & Ahn, ; Rodriguez‐Feo, Gallego‐Delgado, Puerto, Wandosell, & Osende, ; Surma et al, ; van Vliet et al, ). Although some dinoflagellates such as Pyrocystis lunula have well‐defined F‐actin filaments (Heimann et al, ), the actin cytoskeleton of L. polyedra is more similar to that of Symbiodinium kawagutii , which has diffuse semi‐structured F‐actin (Villanueva et al, ). It is challenging to quantify F‐actin in L. polyedra without more information about the efficiency of phalloidin binding to F‐actin.…”

“…Cytoskeletal features can influence mechanosensitivity by acting as a scaffold‐like structure that sensitizes or desensitizes cells to stress‐induced events depending on the organism and the system. Two of the main types of cytoskeletal scaffold structures are microtubules and F‐actin (Pradel, Santini, Bernadac, Fukumori, & Wu, ; Villanueva, Arzápalo‐Castañeda, & Castillo‐Medina, ; Walsh, ).…”

“…Dinoflagellates possess a well‐organized microtubule cytoskeleton that consists of bands of longitudinal microtubules that run from the apical and antapical poles of the cell to the cingulum, a groove around the “equator” of the cell that houses the transverse flagellum, that also contains microtubule bundles (Brown et al, ; Hansen & Moestrup, ; Roberts & Roberts, ; Roberts, Lemoine, Schneider, & Farmer, ; Sekida, Takahira, Horiguchi, & Okuda, ; Villanueva et al, ; von Dassow et al, ; von Dassow, ). The sulcus, a groove on the ventral surface that runs from the cingulum to the antapical end of the cell, houses the longitudinal flagellum of the cell.…”

“…The sulcus, a groove on the ventral surface that runs from the cingulum to the antapical end of the cell, houses the longitudinal flagellum of the cell. The dinoflagellate cortical F‐actin cytoskeleton is more diffuse (Roberts & Roberts, ; Soyer‐Gobillard, Ausseil, & Géraud, ; Villanueva et al, ), although members of the genus Pyrocystis have an extensive well‐defined actin network (Heimann, Klerks, & Hasenstein, ).…”