ABSTRACT
The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.
Subject(s)
Albumins/metabolism , Golgi Apparatus/metabolism , alpha 1-Antitrypsin/metabolism , Biological Transport , Computer Simulation , Diffusion , Endoplasmic Reticulum/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Hep G2 Cells , Humans , Light , Microscopy, Confocal , Microscopy, Immunoelectron , Microscopy, Video , Protein TransportABSTRACT
Gold structures can be created in a scanning electron microscope (SEM) from the Me(2)Au(acac) precursor by direct writing with the electron beam. The as-deposited purity is usually poor, and a common purification approach is a post-annealing step that indeed is effective but also induces a volume reduction because of carbon loss and an undesirable reconfiguration of the gold structure, resulting in the loss of the original shape. We studied the shape change as a result of such purification, and to minimize this effect, the application of a tantalum and chromium buffer layer was investigated. These buffer materials are well-known for their good adhesion properties. We confirm by dedicated SEM, atomic force microscopy (AFM), and transmission electron microscopy (TEM) analysis that, for the creation of a uniform Au structure, tantalum is a better buffer layer material than chromium. Post-annealing of the Au electron-beam-induced deposition (EBID) patterns for 1 h at 600 °C in air resulted in a dramatic purity increase (from 8-12 atomic % Au to above 92 atomic % Au). The uncovered part of the tantalum layer can be easily etched away, resulting in a well-defined, high-purity, gold structure.
