BN Seminar - Francesca Bottanelli (Yale University): 'Live-cell nanoscopy of protein sorting at the Golgi'



14:00 hrs


A1.100, TU Delft


The Golgi apparatus is the main sorting station of the cell and despite decades of research we are still far from fully understanding how it functions. 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. Progress has been hampered by 1) the difficulty to image molecular processes in the intrinsically crowded perinuclear area using standard diffraction limited imaging techniques and 2) the possibility of introducing artifacts when important machinery components are over-expressed.

We have recently developed a novel labeling strategy for dual-color live-cell stimulated emission depletion (STED) super-resolution imaging. Previous demonstrations of multi-color live-cell STED had failed to translate into widespread application due to limitations of the available fluorophores (insufficient photostability, lack of cell permeability, crosstalk). Here we report that SNAP and Halo substrates of the dye ATTO590 can cross biological membranes to provide a second color needed to complement the well known live-cell STED-compatible dye silicone rhodamine (SiR).

Taking advantage of gene editing techniques (CRISPR/Cas9) to avoid over-expression and our novel live-cell STED labeling strategy we revisit the role of ARF1. Small GTPases of the ARF family are the main molecular regulators of the secretory pathway. The role of ARF is mainly to recruit adaptors and coat proteins for the formation of transport carriers. Beside its well-established role in generating COPI vesicles by recruiting Coatomer at the Golgi, we find that ARF1 is additionally involved in the formation of anterograde and retrograde tubular carriers. The ARF1-positive tubular carriers can be divided into two subclasses: i) anterograde tubules that are also positive for Clathrin and contain the cargo VSV G, and ii) retrograde tubules that are positive for Coatomer and contain the cargo KDEL receptor. ARF1 tubular transport intermediates account for a major membrane flow out of the Golgi.