Structure and Protein Composition of a Basal‐Body Scaffold (“Cage”) in the Hypotrich Ciliate Euplotes

Abstract

Cilia on the ventral surface of the hypotrich ciliate Euplotes are clustered into polykinetids or compound ciliary organelles, such as cirri or oral membranelles, used in locomotion and prey capture. A single polykinetid may contain more than 150 individual cilia; these emerge from basal bodies held in a closely spaced array within a scaffold or framework structure that has been referred to as a basal-body "cage". Cage structures were isolated free of cilia and basal bodies; the predominant component of such cages was found on polyacrylamide gels to be a 45-kDa polypeptide. Antisera were raised against this protein band and used for immunolocalizations at the light and electron microscope levels. Indirect immunofluorescence revealed the 45-kDa polypeptide to be localized exclusively to the bases of the ventral polykinetids. Immunogold staining of thin sections of intact cells further localized this reactivity to filaments of a double-layered dense lattice that appears to link adjoining basal bodies into ordered arrays within each polykinetid. Scanning electron microscopy of isolated cages reveals the lower or "basal" cage layer to be a fine lacey meshwork supporting the basal bodies at their proximal ends; adjoining basal bodies are held at their characteristic spacing by filaments of an upper or "medial" cage layer. The isolated cage thus resembles a miniature test-tube rack, able to accommodate varying arrangements of basal-body rows, depending on the particular type of polykinetid. Because of its clear and specific localization to the basal-body cages in Euplotes, we have termed this novel 45-kDa protein "cagein".

Sequence and Properties of Cagein, a Coiled‐Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotesaediculatus

“…Euplotes cell culture, cortical preparations, and antibody production Previous publications have described in detail the methods used to raise Euplotes aediculatus, prepare purified cortical extracts from them, and raise antibodies against select cortical proteins (Kloetzel 1991;Kloetzel et al 2003a;Kloetzel and Brann 2012). In brief, E. aediculatus (ATCC 30859) were cultivated in modified Pringsheim's medium, or in commercial Volvic water, and fed either bacteria or Tetrahymena.…”

“…Extraction of cortical fractions from large quantities of washed Euplotes cells utilized a Triton-high salt (THS) protocol modified slightly from that published by Williams et al (1989). Protein bands from THS-extracted cortical preparations were separated by conventional SDS-PAGE and electroblotted onto nitrocellulose membranes; polyclonal antibodies were then produced in rabbits immunized with individual bands cut from the nitrocellulose, as described (Kloetzel and Brann 2012).…”

“…Since the amino acid heptads involved in coiled-coil formation are rich in charged residues (Mason and Arndt 2004), this may help explain the high percentage of charged amino acids cited above in cagein's composition. Cytoskeletal proteins often possess significant domains of coiled-coil structure (see discussion in Preisner et al 2018); thus, it seems reasonable to interpret cagein as an intermediatefilament (IF) type protein that assembles into "cage" scaffolds, grouping basal-body arrays in the Euplotes cortex (Kloetzel and Brann 2012). The potential for serine, threonine, and tyrosine (STY) residues in the cagein protein to serve as substrates for protein phosphorylation was explored using the Net-Phos3.1a program (ExPASy/CBS).…”

“…Euplotids lack, in the mature structures, prominent striated kinetodesmal fibers (rootlets) and related appendages found in most ciliates (see Lynn and Corliss 1991); their cage structures thus may provide additional support and anchoring against the mechanical stresses of ciliary activity. Since basal-body cages can be isolated as intact "organelles," free of basal bodies (Kloetzel and Brann 2012), additional proteins may be presumed to play roles as the "glue" anchoring the many individual kinetosomes into the cage frameworks. Such proteins may be related to those already identified as kinetosome-associated in earlier investigations of the ciliate cortex (Heydeck et al 2016;Kilburn et al 2007) and other systems (Jana et al 2018).…”

Morpholino-Mediated Knockdown of Ciliary Genes in Euplotes vannus, a Novel Marine Ciliated Model Organism

“…Euplotid ciliates, represented by Euplotes vannus, the focus of this work, are the most highly differentiated and complicated group of ciliates. Euplotid cells differentiate into dorsal-ventral sides, and their cilia are diversified into different types, including cirri on the ventral side, which are composed of dozens or 100s of cilia joined together to form a tuft that functions as a "leg"; adoral zone membranelles, which are composed of rows of cilia that are fused together to form multiple membranelles, whose constant beating are vital for food absorption and function as a "mouth"; and single shorter cilia on the dorsal side, which are arranged in multiple rows and may possess sensory functions (Lynn, 2008;Jiang et al, 2010;Kloetzel and Brann, 2012). In addition, euplotids have many other intriguing and unique biological features.…”