multimodal-hierarchical-imaging-serial-sections-for-finding-specific

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Universität Heidelberg

Bloodstream-form Trypanosoma brucei have two 14-3-3 proteins, which are required for parasite multiplication. We here describe the effects of 14-3-3 depletion on vesicular transport of variant surface glycoprotein (VSG). 14-3-3 depletion had no detectable effect on de novo synthesis and trafficking of VSG to the cell surface, or on VSG endocytosis. Despite strong inhibition of cell division, the flagellar pocket was not enlarged and the ultrastructure of internal organelles appeared normal. The Rab11-positive recycling endosome compartment was, however, fivefold smaller than normal, and the rate of return of recycling VSG to the surface was correspondingly reduced. Down-regulating 14-3-3 also prevented enlargement of the flagellar pocket by clathrin depletion. These results suggest that there is a remarkably specific requirement for 14-3-3 in normal functioning of the Rab11-positive recycling endosome compartment.

Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling.

Here we describe refinements in the processing of high-pressure frozen samples of delicate plant tissues for immuno-electron microscopy. These involve: shortened freeze-substitution schedules, lower temperatures during processing and polymerisation, the avoidance of temperature fluctuations and the optimisation of heat transfer from the specimens using small disposable aluminium containers. The application of these modifications leads to very good structural preservation and selective membrane contrast. As a result, the versatility of the method is increased since not only immuno-electron microscopical studies can be performed but often the quality is also quite suitable for structural investigations.

An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast.

Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200 nm. The size distribution revealed that endocytotic vesicles with a median at about 132 nm were smaller than exocytotic vesicles with a median at 155 nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.

High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast.

Imaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips.

Article	Year
Depletion of 14-3-3 proteins in bloodstream-form Trypanosoma brucei inhibits variant surface glycoprotein recycling. International journal for parasitology\| PubMed ID: 19925803	2010
An improved procedure for low-temperature embedding of high-pressure frozen and freeze-substituted plant tissues resulting in excellent structural preservation and contrast. Journal of microscopy\| PubMed ID: 22360578	2012
High-resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast. Traffic (Copenhagen, Denmark)\| PubMed ID: 25712715	2015
Hierarchical imaging: a new concept for targeted imaging of large volumes from cells to tissues. BMC cell biology\| PubMed ID: 27955619	2016

Stefan Hillmer

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