Further investigation to the role of Arf1 in the Golgi apparatus

Speaker: Francesca Bottanelli
Department:
Yale University
Subject: Live-cell nanoscopy of protein sorting at the Golgi
Location: A1.100 (TU Delft)
Date: 14-11-2016

Kristian Blom

At the 14th of November I visited a BN seminar given by dr. Francesca Bottanelli, a postdoctoral associate in cell biology in the Rothman Lab at Yale University. Dr. Bottanelli is a cell biologist who is interested in the use of Stimulated Emission Depletion (STED) for living cell imaging. At the moment she uses STED to image COPI protein dynamics at the Golgi apparatus in mammalian cells. 

Dr. Bottanelli started the talk by introducing the main actor of her research: the Golgi apparatus. This organelle is the main sorting station of the cell and despite decades of research we are still far from fully understanding how it functions. Proteins that are synthesized in the endoplasmic reticulum are packaged into vesicles, which then fuse with the Golgi apparatus. Thereafter they are transported to the right location in the cell. While the machinery and molecular interactions involved in cargo sorting have been extensively investigated in vitro, there is a lack of understanding of the dynamics and nanoscale organization in living cells, since intracellular transport occurs over extremely short distances of a few 100 nm. A little progress is made over the past decade due to the difficulty to image molecular processes in the intrinsically crowded perinuclear area using standard diffraction limited imaging techniques.

After the introduction we went further into detail about STED. This imaging technique is part of the so called super-resolution microscope techniques, which means that the resolution that you can get with this technique bypasses the diffraction limit. In conventional fluorescence microscopy the electrons of a fluorophore are excited by incoming light of a certain wavelength, and thereafter by relaxation of the excited electron light of one specific wavelength is emitted back by the fluorophore (green arrow figure 1). However, with STED it is possible to force the excited electron into a higher relaxed vibration state than the fluorescence transition would enter, causing the released photon to be red-shifted (orange arrow figure 1). To make this alternative emission occur, an incident photon must strike the fluorophore. One can achieve super resolution by depleting fluorescence in specific regions of the sample while leaving a center focal spot active to emit fluorescence.

Figure 1 – A Jablonski diagram for the process of fluorescence and STED. Blue: Excitation of an fluorophore’s electron to a higher energy state. Green: Fluorescence – Relaxation of the excited electron. Due to the drop in energy light is emitted. Orange: STED – Relaxation of the excited electron to a higher energy state compared to the orange version. Due to the smaller energy drop during relaxation, light with a longer wavelength is emitted.

Dr. Bottanelli developed a novel labeling strategy for dual-color live-cell STED. Taking advantage of gene editing techniques (CRISPR/Cas9) and her novel live-cell STED labeling strategy she took a careful look at the role of ARF1, a protein localized in the Golgi apparatus that has a central role in the formation of COPI vesicles at the Golgi. The role of ARF is mainly to recruit adaptors and coat proteins for the formation of transport carriers. However, Dr. Botanelli found out that besides its role in generating COPI vesicles by recruiting Coatomer at the the Golgi, ARF1 also is involved in the formation of anterograde (from ER to Golgi) and retrograde (from Golgi to ER) tubular carriers.

Due to my lack of knowledge about the Golgi apparatus and the different proteins that are involved in the transportation process, it was rather hard for me to follow the talk. However, once I started to put everything on paper, more and more about what was told became clear for me. Afterwards I asked myself the question whether dr. Botanelli already found out if these anterograde and retrograde carriers are the main transportation systems that allow for the protein dynamics between the ER and Golgi apparatus. Since I did not followed everything during the talk, it could be that I missed that part. If that is not the case it might be a nice topic to do research about for in the future.

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