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SummaryProteins synthesised at the endoplasmic reticulum (ER) have to undergo a number of consecutive and coordinated steps to reach the Golgi complex. To understand the dynamic complexity of ER-to-Golgi transport at the structural and molecular level, light microscopy approaches are fundamental tools that allow in vivo observations of protein dynamics and interactions of fluorescent proteins in living cells. Imaging protein and organelle dynamics close to the ultra-structural level became possible by combining light microscopy with electron microscopy analyses or super-resolution light microscopy methods. Besides, increasing evidence suggests that the early secretory pathway is tightly connected to other cellular processes, such as signal transduction, and quantitative information at the systems level is fundamental to achieve a comprehensive molecular understanding of these connections. High-throughput microscopy in fixed and living cells in combination with systematic perturbation of gene expression by, e.g. RNA interference, will open new avenues to gain such an understanding of the early secretory pathway at the systems level. In this Commentary, we first outline examples that revealed the dynamic organisation of ER-to-Golgi transport in living cells. Next, we discuss the use of advanced imaging methods in studying ER-to-Golgi transport and, finally, delineate the efforts in understanding ER-to-Golgi transport at the systems level.

Summary The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes [1–3]. Both stimulate the GTPase activity of Sar1p [4], which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd.

We present an approach for the quantification of fluorescent spots in time series of 3-D confocal microscopy images of endoplasmic reticulum exit sites of dividing cells. Fluorescent spots are detected based on extracted image regions of highest response using the HMAX transform and prior convolution of the 3-D images with a Gaussian kernel. The sensitivity of the involved parameters was studied and a quantitative evaluation using both 3-D synthetic and 3-D real data was performed. The approach was successfully applied to more than one thousand 3-D confocal microscopy images.

Biosynthetic cargo enters the secretory pathway at the endoplasmic reticu-lum (ER) where it is properly folded before it becomes sorted and concentrated into transport carriers that move towards the Golgi complex. The basic protein machinery involved in bidirectional transport between the ER and the Golgi complex has long been identified and is fairly well characterized (Lee et al. 2004). The vesicular coat complex COPII mediates ER exit, while retrograde transport from the Golgi complex to the ER involves the function of the vesicular coat complex COPI. In the following paragraphs we focus on our current knowledge on the spatial and temporal organization of ER-to-Golgi transport at the molecular level.