Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods.

backgroundCilia are vital to various cellular and sensory functions. The pathway by which ciliary membrane proteins translocate through the transition zone is not well understood. Direct morphological characterization of ciliary cargoes in transit remains lacking. In the vertebrate photoreceptor, rhodopsin is synthesized and transported from the inner segment to the disc membranes of the outer segment (OS), which is a modified cilium. To date, the membrane topology of the basal OS and the mechanisms by which rhodopsin is transported through the transition zone (i.e., connecting cilium) and by which nascent disc membranes are formed remain controversial.

resultsUsing an antibody recognizing its cytoplasmic C-terminus, we localize rhodopsin on both the plasma membrane and lumen of the connecting cilium by immuno-electron microscopy (EM). We also use transmission EM to visualize the electron-dense enzymatic products derived from the rhodopsin-horseradish peroxidase (HRP) fusion in transfected rodent rods. In the connecting cilium, rhodopsin is not only expressed in the plasma membrane but also in the lumen on two types of membranous carriers, long smooth tubules and small, coated, filament-bound vesicles. Additionally, membrane-bound rhodopsin carriers are also found in close proximity to the nascent discs at the basal OS axoneme and in the distal inner segment. This topology-indicative HRP-rhodopsin reporter shows that the nascent basalmost discs and the mature discs have the same membrane topology, with no indication of evagination or invagination from the basal OS plasma membranes. Serial block face and focus ion beam scanning EM analyses both indicate that the transport carriers enter the connecting cilium lumen from either the basal body lumen or cytoplasmic space between the axonemal microtubules and the ciliary plasma membrane.

conclusionsOur results suggest the existence of multiple ciliary gate entry pathways in rod photoreceptors. Rhodopsin is likely transported across the connecting cilium on the plasma membrane and through the lumens on two types of tubulovesicular carriers produced in the inner segment. Our findings agree with a previous model that rhodopsin carriers derived from the cell body may fuse directly onto nascent discs as they grow and mature.