application-high-speed-super-resolution-speed-microscopy-live-primary

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

The primary cilium is a microtubule-based protrusion on the surface of many eukaryotic cells and contains a unique complement of proteins that function ...

Temple University

O-GlcNAc-ylation is the post-translational addition of an O-linked β-N-acetylglucosamine to the serine and threonine residues of thousands of proteins in eukaryotic cells. Specifically, half of the thirty different types of protein components in the nuclear pore complex (NPC) are modified by O-GlcNAc, of which the majority are intrinsically disordered nucleoporins (Nups) containing multiple phenylalanine-glycine (FG) repeats. Moreover, these FG-Nups form a strict selectivity barrier with a high density of O-GlcNAc in the NPC to mediate bidirectional trafficking between the cytoplasm and nucleus. However, the roles that O-GlcNAc plays in the structure and function of the NPC remain obscure. In this review paper, we will discuss the current knowledge of O-GlcNAc-ylated Nups, highlight some new techniques used to probe O-GlcNAc's roles in the nuclear pore, and finally propose a new model for the effect of O-GlcNAc on the NPC's permeability.

O-GlcNAc-ylation in the Nuclear Pore Complex.

Transport of membrane and cytosolic proteins in primary cilia is thought to depend on intraflagellar transport (IFT) and diffusion. However, the relative contribution and spatial routes of each transport mechanism are largely unknown. Although challenging to decipher, the details of these routes are essential for our understanding of protein transport in primary cilia, a critically affected process in many genetic diseases. By using a high-speed virtual 3D super-resolution microscopy, we have mapped the 3D spatial locations of transport routes for various cytosolic proteins in the 250-nm-wide shaft of live primary cilia with a spatiotemporal resolution of 2 ms and <16 nm. Our data reveal two spatially distinguishable transport routes for cytosolic proteins: an IFT-dependent path along the axoneme, and a passive-diffusion route in the axonemal lumen that escaped previous studies. While all cytosolic proteins tested primarily utilize the IFT path in the anterograde direction, differences are observed in the retrograde direction where IFT20 only utilizes IFT, and approximately half of KIF17 and one third of α-tubulin utilizes diffusion besides IFT.

Andrew Ruba

Department of Biology

Temple University

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

Andrew Ruba

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Temple University

Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium

Department of Biology