244 research outputs found
FAPP2 is involved in the transport of apical cargo in polarized MDCK cells
Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells
Using size-selected gold clusters on graphene oxide films to aid cryo-transmission electron tomography alignment
A three-dimensional reconstruction of a nano-scale aqueous object can be achieved by taking a series of transmission electron micrographs tilted at different angles in vitreous ice: cryo-Transmission Electron Tomography. Presented here is a novel method of fine alignment for the tilt series. Size-selected gold clusters of ~2.7 nm (Au(561 ± 14)), ~3.2 nm (Au(923 ± 22)), and ~4.3 nm (Au(2057 ± 45)) in diameter were deposited onto separate graphene oxide films overlaying holes on amorphous carbon grids. After plunge freezing and subsequent transfer to cryo-Transmission Electron Tomography, the resulting tomograms have excellent (de-)focus and alignment properties during automatic acquisition. Fine alignment is accurate when the evenly distributed 3.2 nm gold particles are used as fiducial markers, demonstrated with a reconstruction of a tobacco mosaic virus. Using a graphene oxide film means the fiducial markers are not interfering with the ice bound sample and that automated collection is consistent. The use of pre-deposited size-selected clusters means there is no aggregation and a user defined concentration. The size-selected clusters are mono-dispersed and can be produced in a wide size range including 2–5 nm in diameter. The use of size-selected clusters on a graphene oxide films represents a significant technical advance for 3D cryo-electron microscopy
Correlative light electron microscopy using small gold nanoparticles as single probes
Correlative light electron microscopy (CLEM) requires the availability of
robust probes which are visible both in light and electron microscopy. Here we
demonstrate a CLEM approach using small gold nanoparticles as a single probe.
Individual gold nanoparticles bound to the epidermal growth factor protein were
located with nanometric precision background-free in human cancer cells by
light microscopy using resonant four-wave-mixing (FWM), and were correlatively
mapped with high accuracy to the corresponding transmission electron microscopy
images. We used nanoparticles of 10 nm and 5 nm radius, and show a correlation
accuracy below 60 nm over an area larger than 10 um size, without the need for
additional fiducial markers. Correlation accuracy was improved to below 40 nm
by reducing systematic errors, while the localisation precision is below 10 nm.
Polarisation-resolved FWM correlates with nanoparticle shapes, promising for
multiplexing by shape recognition in future applications. Owing to the
photostability of gold nanoparticles and the applicability of FWM microscopy to
living cells, FWM-CLEM opens up a powerful alternative to fluorescence-based
methods
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