Quantitative intra-Golgi transport and organization data suggest the stable compartment nature of the Golgi

Reviewer #1 (Public Review):

Summary:

In the manuscript by Tie et.al., the authors couple the methodology which they have developed to measure LQ (localization quotient) of proteins within the Golgi apparatus along with RUSH based cargo release to quantify the speed of different cargos traveling through Golgi stacks in nocodazole induced Golgi ministacks to differentiate between cisternal progression vs stable compartment model of the Golgi apparatus. The debate between cisternal progression model and stable compartment model has been intense and going on for decades and important to understand the basic way of function/organization of the Golgi apparatus. As per the stable compartment model, cisterna are stable structures and cargo moves along the Golgi apparatus in vesicular carriers. While as per cisternal progression model, Golgi cisterna themselves mature acquiring new identity from the cis face to the trans face and act as transport carriers themselves. In this work, authors provide a missing part regarding intra-Golgi speed for transport of different cargoes as well as the speed of TGN exit and based on the differences in the transport velocities for different cargoes tested favor a stable compartment model. The argument which authors make is that if there is cisternal progression, all the cargoes should have a similar intra-Golgi transport speed which is essentially the rate at which the Golgi cisterna mature. Furthermore, using a combination of BFA and Nocodazole treatments authors show that the compartments remain stable in cells for at least 30-60 minutes after BFA treatment.

Strengths:

The method to accurately measure localization of a protein within the Golgi stack is rigorously tested in the previous publications from the same authors and in combination with pulse chase approaches has been used to quantify transport velocities of cargoes through the Golgi. This is a novel aspect in this paper and differences in intra-Golgi velocities for different cargoes tested makes a case for a stable compartment model.

Weaknesses:

Experiments are only tested in one cell line (HeLa cells) and predominantly derived from experimental paradigm using RUSH assays where a secretory cargo is released in a wave (not the most physiological condition) and therefore additional approaches would make a more compelling case for the model.

Reviewer #2 (Public Review):

Summary:

This manuscript describes the use of quantitative imaging approaches, which have been a key element of the labs work over the past years, to address one of the major unresolved discussions in trafficking: intra-Golgi transport. The approach used has been clearly described in the labs previous papers, and is thus clearly described. The authors clearly address the weaknesses in this manuscript and do not overstate the conclusions drawn from the data. The only weakness not addressed is the concept of blocking COPI transport with BFA, which is a strong inhibitor and causes general disruption of the system. This is an interesting element of the paper, which I think could be improved upon by using more specific COPI inhibitors instead, although I understand that this is not necessarily straightforward.

I commend the authors on their clear and precise presentation of this body of work, incorporating mathematical modelling with a fundamental question in cell biology. In all, I think that this is a very robust body of work, that provides a sound conclusion in support of the stable compartment model for the Golgi.

General points:

The manuscript contains a lot of background in its results sections, and the authors may wish to consider rebalancing the text: The section beginning at Line 175 is about 90% background and 10% data. Could some data currently in supplementary be included here to redress this balance, or this part combined with another?

Reviewer #3 (Public Review):

The manuscript by Tie et al. provides a quantitative assessment of intra-Golgi transport of diverse cargos. Quantitative approaches using fluorescence microscopy of RUSH synchronized cargos, namely GLIM and measurement of Golgi residence time, previously developed by the author's team (publications from 20216 to 2022), are being used here.

Most of the results have been already published by the same team in 2016, 2017, 2020 and 2021. In this manuscript, very few new data have been added. The authors have put together measurements of intra-Golgi transport kinetics and Golgi residence time of many cargos. The quantitative results are supported by a large number of Golgi mini-stacks/cells analyzed. They are discussed with regard to the intra-Golgi transport models being debated in the field, namely the cisternal maturation/progression model and the stable compartments model. However, over the past decades, the cisternal progression model has been mostly accepted thanks to many experimental data.

The authors show that different cargos have distinct intra-Golgi transport kinetics and that the Golgi residence time of glycosyltransferases is high. From this and the experiment using brefeldinA, the authors suggest that the rim progression model, adapted from the stable compartments model, fits with their experimental data.

Strengths:

The major strength of this manuscript is to put together many quantitative results that the authors previously obtained and to discuss them to give food for thought about the intra-Golgi transport mechanism.
The analysis by fluorescence microscopy of intra-Golgi transport is tough and is a tour de force of the authors even if their approach show limitations, which are clearly stated. Their work is remarkable in regards to the numbers of Golgi markers and secretory cargos which have been analyzed.

Weaknesses:

As previously mentioned, most of the data provided here were already published and thus accessible for the community. Is there is a need to publish them again?
The authors' discussion about the intra-Golgi transport model is rather simplistic. In the introduction, there is no mention of the most recent models, namely the rapid partitioning and the rim progression models. To my opinion, the tubular connections between cisternae and the diffusion/biochemical properties of cargos are not enough taken into account to interpret the results. Indeed, tubular connections and biochemical properties of the cargos may affect their transit through the Golgi and the kinetics with which they reach the TGN for Golgi exit.
Nocodazole is being used to form Golgi mini-stacks, which are necessary to allow intra-Golgi measurement. The use of nocodazole might affect cellular homeostasis but this is clearly stated by the authors and is acceptable as we need to perturb the system to conduct this analysis. However, the manual selection of the Golgi mini-stack being analyzed raises a major concern. As far as I understood, the authors select the mini-stacks where the cargo and the Golgi reference markers are clearly detectable and separated, which might introduce a bias in the analysis.
The terms 'Golgi residence time ' is being used but it corresponds to the residence time in the trans-cisterna only as the cargo has been accumulated in the trans-Golgi thanks to a 20{degree sign}C block. The kinetics of disappearance of the protein of interest is then monitored after 20{degree sign}C to 37{degree sign}C switch.
Another concern also lies in the differences that would be introduced by different expression levels of the cargo on the kinetics of their intra-Golgi transport and of their packaging into post-Golgi carriers.