Immunolocalization of Actin in Paramecium Cells

Abstract

S U M M A R YWe have selected a conserved immunogenic region from several actin genes of Paramecium , recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several m in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including "discoidal" vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally welldefined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any largescale redistribution during preparation. A ctin is a highly flexible cytoskeletal component that participates in many static and dynamic functions in eukaryotic cells (Pollard et al. 2000). This includes reversible self-assembly of monomeric G-actin to F-actin filaments. Also generally known is that these filaments may be more or less bundled and can serve different functions, such as structural enforcement and restructuring of the cell cortex, rearrangement of cortical components during intracellular signaling, organelle dynamics and transport, etc. The latter includes wellestablished functions such as phagosome formation and plasma streaming, i.e., cyclosis (Shimmen and Yokota 2004). However, quite recent results highlight a much broader functional spectrum of F-actin than previously assumed. This applies to early steps of exocytosis, including dense core vesicle docking (Morales et al. 2000;Pendleton and Koffer 2001;Manneville et al. 2003;Gasman et al. 2004), late steps of endocytosis (Engqvist-Goldstein and Drubin 2003;Guilherme et al. 2004), exo-endocytosis coupling (Valentijn et al. 1999), endo-phagosome interaction (Kjeken et al. 2004), delivery of endocytosed receptors to lysosomes for degradation (Stoorvogel et al. 2004), vacuole fusion in yeast (Merz and Wickner 2004), and positioning of the nucleus (Starr and Han 2003). Some aspects are still poorly understood, particularly, e.g., the role of actin in flagella of algae (Mitchell 2000;Hayashi et al. 2001;Hirono et al. 2003), whereas its occurrence in cilia has remained a matter of debate. Another line of experiments concerns the potential role of actin in mediating the connection between cortical Ca 2 ϩ -stores and the plasma membrane...

A broad spectrum of actin paralogs inParamecium tetraureliacells display differential localization and function

“…Comparison with previous localization studies Previously we localized actin in Paramecium by using fluorescently tagged DNase, heavy meromyosin, phalloidin and anti-actin antibodies (Tiggemann and Plattner, 1981;Kersken et al, 1986a;Kersken et al, 1986b) and, more specifically, using anti-actin1-1 antibodies (Kissmehl et al, 2004). Altogether this resulted in labeling of cilia, basal bodies, Journal of Cell Science 120 (1) Fig.…”

“…The region chosen has less than 25% identity to other subfamilies, which makes it unlikely that the antibodies will react with any of the other actins or ARPs. The antibodies were affinity purified not only against the peptide for immunization, but also against peptides used to raise antibodies against act1-1 ( Kissmehl et al, 2004) and against act5-1, and were tested in western blots for their crossreactivity, which was negative (Fig. 4A).…”

“…*As the actin isoforms act1-1, act1-2 and act1-3 are identical at the amino acid level (I.M.S., J.M., C.R., E. Wagner, H.P. and R.K., unpublished), the polyclonal antibodies against act1-1 will also recognize those isoforms and probably also peptides from further members of the act1 subfamily and possibly also of other subfamilies (Kissmehl et al, 2004).…”

“…We considered that cytochalasin B treatment abolished trichocyst docking in Paramecium (Beisson and Rossignol, 1975) and the occurrence of anti-actin antibody labeling around trichocysts docked at the plasma membrane (Kissmehl et al, 2004). With ongoing cell divisions, every daughter cell has to produce a new arsenal of trichocysts and to transport them to the cell cortex.…”

Is it possible to consider «kinetomics» and «argyromics» as novel trends of functional morphology and systems biology of parasitic and nonparasitic protozoa? a brief analytical review

“…This technique provides a rational basis on which the morphologists can make conclusions about different cell components. This approach can be extended by the simple general-biological interpolations to the Protozoa morphology not only because immunofluorescence staining methods are applicable for protistological / protozoological studies from 1960 till now (Zaman, 1965;Zaman, 1966;Dzbenski, 1966;Hauser, 1980;Kovac, 1980;Ohba, 1986;Brugerolle, 1988;Olins, 1989;Allen, 1990;Turkewitz, 1992;Dohra, 1994;Hanyu, 1995;Hanyu, 1996;Clerot, 2001;Santangelo, 2001;Strzyzewska-Jówko, 2003;McLaughlin, 2004;Itabashi, 2004;Kissmehl, 2004;Ramoino, 2004), but also because the simplest inorganic staining agents may be used for visualization of several compartments and constituents of the Protozoa cells (even without the antibodyome agents (Mohebbi, 2009;Zhu, 2013)). Notwithstanding, there is no case of determination of the different "omics" characteristics using inorganic dyes.…”