Epiplasts: Membrane Skeletons and Epiplastin Proteins in Euglenids, Glaucophytes, Cryptophytes, Ciliates, Dinoflagellates, and Apicomplexans

ABSTRACTAnimals and amoebae assemble actin/spectrin-based plasma membrane skeletons, forming what is often called the cell cortex, whereas euglenids and alveolates (ciliates, dinoflagellates, and apicomplexans) have been shown to assemble a thin, viscoelastic, actin/spectrin-free membrane skeleton, here called the epiplast. Epiplasts include a class of proteins, here called the epiplastins, with a head/medial/tail domain organization, whose medial domains have been characterized in previous studies by their low-complexity amino acid composition. We have identified two additional features of the medial domains: a strong enrichment of acid/base amino acid dyads and a predicted β-strand/random coil secondary structure. These features have served to identify members in two additional unicellular eukaryotic radiations—the glaucophytes and cryptophytes—as well as additional members in the alveolates and euglenids. We have analyzed the amino acid composition and domain structure of 219 epiplastin sequences and have used quick-freeze deep-etch electron microscopy to visualize the epiplasts of glaucophytes and cryptophytes. We define epiplastins as proteins encoded in organisms that assemble epiplasts, but epiplastin-like proteins, of unknown function, are also encoded in Insecta, Basidiomycetes, andCaulobactergenomes. We discuss the diverse cellular traits that are supported by epiplasts and propose evolutionary scenarios that are consonant with their distribution in extant eukaryotes.IMPORTANCEMembrane skeletons associate with the inner surface of the plasma membrane to provide support for the fragile lipid bilayer and an elastic framework for the cell itself. Several radiations, including animals, organize such skeletons using actin/spectrin proteins, but four major radiations of eukaryotic unicellular organisms, including disease-causing parasites such asPlasmodium, have been known to construct an alternative and essential skeleton (the epiplast) using a class of proteins that we term epiplastins. We have identified epiplastins in two additional radiations and present images of their epiplasts using electron microscopy. We analyze the sequences and secondary structure of 219 epiplastins and present an in-depth overview and analysis of their known and posited roles in cellular organization and parasite infection. An understanding of epiplast assembly may suggest therapeutic approaches to combat infectious agents such asPlasmodiumas well as approaches to the engineering of useful viscoelastic biofilms.

A malaria membrane skeletal protein is essential for normal morphogenesis, motility, and infectivity of sporozoites

Membrane skeletons are structural elements that provide mechanical support to the plasma membrane and define cell shape. Here, we identify and characterize a putative protein component of the membrane skeleton of the malaria parasite. The protein, named PbIMC1a, is the structural orthologue of the Toxoplasma gondii inner membrane complex protein 1 (TgIMC1), a component of the membrane skeleton in tachyzoites. Using targeted gene disruption in the rodent malaria species Plasmodium berghei, we show that PbIMC1a is involved in sporozoite development, is necessary for providing normal sporozoite cell shape and mechanical stability, and is essential for sporozoite infectivity in insect and vertebrate hosts. Knockout of PbIMC1a protein expression reduces, but does not abolish, sporozoite gliding locomotion. We identify a family of proteins related to PbIMC1a in Plasmodium and other apicomplexan parasites. These results provide new functional insight in the role of membrane skeletons in apicomplexan parasite biology.

Cryoelectron Microscopy of Red Cell Skeletons Reveals that Spectrin has a Helical Conformation

In vivo the red blood cell reversibly and rapidly deforms as it passes through the small capillaries. Although the physiochemical basis of the red cell's deformability is unknown, it is regarded as being a property of the membrane protein spectrin. We have used cryoelectron microscopy to study the structure of spectrin in membrane skeletons to determine how its molecular structure confers elastic properties on the cell membrane. Cryomicroscopy has the virtue of minimally perturbing easily deformable structures such as spectrin.Figure 1 is an electron micrograph of a portion of a frozen-hydrated red cell skeleton. The spectrin molecules are indicated by the arrow heads. They cross link the skeletal network by interacting with short rod like segments F-actin (indicated by the arrows) containing about 13 actin molecules. In such micrographs the spectrin appears to have a sinusoidal shape whereas in negatively stained preparations spectrin appears to be straight.We have used the procedure of Margalef in order to determine the three dimensional trajectory of the spectrin molecule.