Background

Chlamydomonas reinhardtii is a single-celled green alga widely used in studying photosynthesis, cilia, chloroplast biology, and the synthesis of biofuels. It is a leading model system in these fields due to the organism’s well-characterized two apical flagella, inexpensive and simple culture conditions, and a fully sequenced haploid genome allowing for excellent genetic studies. Understanding the Chlamydomonas cytoskeleton will play an important role in advancing these areas of research. However, the filamentous actin network of Chlamydomonas reinhardtii remains uncharacterized, partly due to the difficulties in visualizing filaments in this eukaryotic alga.

Recently, there has been some success in detecting filamentous actin in Chlamydomonas. Through the expression of the fluorescently-tagged actin binding peptide, LifeAct, live-cell imaging revealed an actin-based perinuclear structure (Avasthi et al., 2014; Onishi et al., 2016). However, there is increasing evidence that LifeAct alters cell morphology, actin organization, actin-dependent functions, and preferentially labels a subset of actin structures (Courtemanche et al., 2016, Flores et al., 2019). Further, achieving stable expression is difficult in Chlamydomonas due to epigenetic silencing of exogenous genes (Cerutti et al., 1997) and the need to colocalize actin probes with fixed cell organelle markers is essential for understanding the revitalized field of Chlamydomonas actin biology. Thus, an efficient and specific fixed-cell method of actin visualization would lead to significant advancement in the field and offer novel insights into basic actin biology.

Available actin antibodies do not discriminate between monomeric and filamentous actin. It was previously shown that the widely used fluorescent actin probe phalloidin was ineffective in labeling filamentous actin in vegetative Chlamydomonas cells (Harper et al., 1992). However, phalloidin does label an actin-dense structure in gametic cells known as the fertilization tubule (Detmers et al., 1985). By optimizing phalloidin staining conditions, we are now able to visualize the Chlamydomonas actin network with exquisite detail in vegetative cells (Figure 1). Specifically, reducing the staining incubation time from 30 min to 16 min was essential in achieving optimal signal to noise which allowed for detection above Chlamydomonas’s auto-fluorescent chloroplast. Usage of a brighter and more photostable fluorophore, Atto 488, was also critical in obtaining a high signal to noise ratio and uniform labeling. This protocol allows us to understand how cell body actin filaments are distributed in gametic and vegetative cells for the first time. The previously unidentified filamentous actin structures will be crucial in exploring actin-dependent behaviors and how co-expressed actin genes in Chlamydomonas function to regulate cytoskeletal dynamics. Development of these strategies may prove useful for the study of actin filaments in other protists for which actin visualization has also been challenging.


Figure 1. Phalloidin staining in wild-type vegetative Chlamydomonas reinhardtii. A. Representative raw single widefield image of specific filamentous actin staining using our optimized protocol. B. The deconvolved maximum intensity projection of a Z-stack (0.3 μm steps) of the field visualized in A. C. Overlay of brightfield and fluorescence channels with phalloidin signal indicated in green. D. Deconvolved maximum intensity projection of a Z-stack (0.3 μm steps) of a single cell demonstrating effective phalloidin staining. Scale bars = 5 μm.