Defects in diffusion barrier function of ciliary transition zone caused by ciliopathy variations of TMEM218.

Primary cilia are antenna-like structures protruding from the surface of various eukaryotic cells, and have distinct protein compositions in their membranes. This distinct protein composition is maintained by the presence of the transition zone (TZ) at the ciliary base, which acts as a diffusion barrier between the ciliary and plasma membranes. Defects in cilia and the TZ are known to cause a group of disorders collectively called the ciliopathies, which demonstrate a broad spectrum of clinical features, such as perinatally lethal Meckel syndrome (MKS), relatively mild Joubert syndrome (JBTS), and nonsyndromic nephronophthisis (NPHP). Proteins constituting the TZ can be grouped into the MKS and NPHP modules. The MKS module is composed of several transmembrane proteins and three soluble proteins. TMEM218 was recently reported to be mutated in individuals diagnosed as MKS and JBTS. However, little is known about how TMEM218 mutations found in MKS and JBTS affect the functions of cilia. In this study, we found that ciliary membrane proteins were not localized to cilia in TMEM218-knockout cells, indicating impaired barrier function of the TZ. Furthermore, the exogenous expression of JBTS-associated TMEM218 variants but not MKS-associated variants in TMEM218-knockout cells restored the localization of ciliary membrane proteins. In particular, when expressed in TMEM218-knockout cells, the TMEM218(R115H) variant found in JBTS was able to restore the barrier function of cells, whereas the MKS variant TMEM218(R115C) could not. Thus, the severity of symptoms of MKS and JBTS individuals appears to correlate with the degree of their ciliary defects at the cellular level.

Formation of the B9-domain protein complex MKS1-B9D2-B9D1 is essential as a diffusion barrier for ciliary membrane proteins.

Cilia are plasma membrane protrusions that act as cellular antennae and propellers in eukaryotes. To achieve their sensory and motile functions, cilia maintain protein and lipid compositions that are distinct from those of the cell body. The transition zone (TZ) is a specialized region located at the ciliary base, which functions as a barrier separating the interior and exterior of cilia. The TZ comprises a number of transmembrane and soluble proteins. Meckel syndrome (MKS)1, B9 domain (B9D)1/MKS9, and B9D2/MKS10 are soluble TZ proteins that are encoded by causative genes of MKS and have a B9D in common. We here demonstrate the interaction mode of these B9D proteins to be MKS1-B9D2-B9D1 and demonstrate their interdependent localization to the TZ. Phenotypic analyses of MKS1-knockout (KO) and B9D2-KO cells show that the B9D proteins are involved in, although not essential for, normal cilia biogenesis. Rescue experiments of these KO cells show that formation of the B9D protein complex is crucial for creating a diffusion barrier for ciliary membrane proteins.

Spermatozoon of the freshwater rotifer Brachionus calyciflorus (Rotifera, Monogononta): Advances in morphological and ultrastructural studies.

The morphological and ultrastructural features of the spermatozoon in Brachionus calyciflorus are described using light, fluorescence and transmission electron microscopy (TEM). The mature spermatozoon, which appears to be thread-like, is composed of a slightly expanded anterior of cell body region and a flagellum region without acrosome. The cell body region and flagellum region are respectively 16-27µm and 20-33µm in length (n=60). The spermatozoon is characterized by a mass of dense tubular materials, which occupy most of the cell. Some mitochondria are distributed around the nuclear region in the anterior of the cell body region, while in the posterior portion of cell body, the chromatin often contains a single lobated nucleus arranged at the center of cell. The flagellum contains the classic axoneme (9×2+2) and possesses lateral undulating membrane. Mature B. calyciflorus males have no germ cell stages earlier than the spermatids in the testis. TEM examination reveals rigid rods as well as predominant typical spermatozoon in the testis. Observations, based on successive photographs and videos, enabled a first-time recording of the unique inverted movement of the spermatozoon, which indicated that the movement of the spermatozoon is driven by the flagellum. Our study also provides further supplementary insights into the phylogenetic systematics of the Rotifera.