Peer review of: ucleoplasmic signals promote directed transmembrane protein import simultaneously via (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
Mudumbi et al combine a toolbox of advanced microscopy methods aiming to resolve the long-lasting
 debate about the nature of different INM protein groups in the peripheral channels in the NPC and
 their integration within the nuclear pore membrane.
 Overall, I find the manuscript exciting, well written and presented (professionally presented Figures),
 technical sound, and important to be published. I therefore recommend to accept the manuscript for
 publication in Nature Communications after addressing the following minor comments.
 (1) Could the authors please discuss the impact of the mechanical rigidity of INM proteins in the
 revised version of the manuscript? I appreciate the significance of their length/height. However, the
 reaction diffusion dynamics and their hydrodynamic radius will also depend on the mechanical status
 of the molecules.
 (2) I think that the manuscript would be strengthened if the authors could show dynamic data, which
 allows the readership to judge the quality of the measurements. The post-processed data and
 presentation is very well executed but the raw data acquisition is difficult to judge.
 (3) Could the authors please provide statistical significance of the different mobilities as measure by
 smFRAP in Table 1? Table 1 provides standard deviations but no p-values.
 (4) I believe that Nat Com does not allow abbreviations in the abstract.
Reviewer #2 (Remarks to the Author):
(5) Could the authors please rephrase the sentence starting with "Here" (line 87)? The reader could be
 confused that the summary paragraph of the end of the introduction starts at this location, which only
 follows line 130.
 
In this paper Mudumbi et al, show exciting data providing structural evidence how transmembrane
proteins are imported via the Nuclear Pore Complex to the inner nuclear membrane. With their
advanced and unique single-molecule techniques they make an important contribution to the
understanding of the import pathway of several membrane proteins.
The import route that membrane proteins take has been long debated. While the relevance of sorting
signals, RanGTP, and NTRS have all been a subject of debate, the fiercest debates concerned the
transport route through the NPC. First, multiple labs provided support for the idea that most yeast and
human INM proteins use the peripheral channels observed in several EM studies. However, this
transport route through the lateral channels was never directly shown. In addition to this canonical
route, two yeast INM proteins were proposed to take a different route: they use an active import
mechanism which involves the passage of their NTR-bound end of the molecule through the central
channel, while their transmembrane domains and the C-terminus travel the peripheral channels. The
proposed transport route implied that, at least transiently, openings must exist between the space
immediately aligning the pore membrane and the central channel. Only indirect evidence was available
so far to support this transport route. According to many in the field, this proposed transport route
was incompatible with the current structural data, and hence regarded highly unlikely. Another
concern was that it could be an oddity of these two proteins, and/or an oddity of yeast.
In the current manuscript this longstanding debate about where these proteins translocate is
 addressed using suitable and sophisticated single molecule methods developed by Prof Yang and
 coworkers and using a range of native proteins. Five human inner nuclear membrane proteins are
 studied (NET51, NET59, NET99, LBR, Lap2beta). The existence of the peripheral channel as well as
 passage of Net51/59/99 via the peripheral channels is confirmed. The overall dimensions of the NPC
 scaffold and the peripheral channels are is good agreement with EM studies, which provides
 confidence in the accuracy of the SPEED method (which I cannot evaluate in detail). Most exciting is
 the data on LBR and Lap2beta which show that their NLS and disordered regions pass the central
 channel, at the same place as soluble proteins with transportins, while the transmembrane and
 membrane proximal regions pass through the lateral channels. Most convincing are those
 measurements where the LBR proteins is simultaneously tagged on both termini, so that the
 recordings of transport through the lateral channels and the central channel reflects one and the same
 molecule. These measurements provide the first direct evidence that the NPC can accommodate for
 the hotly debated transport route through the central channel. In fact, the studies by Mudumbi et al
 align well with the emerging view that the structure of the NPC is much more dynamic than previously
 thought. In a set of elegant studies the authors additionally show how perturbations of the NPC
 channels or the sorting signals can alter the transport path of a membrane proteins. This is a
 surprising finding that may turn out to have important biological implications. Also, the authors
 provide kinetic information about both transport routes, which is roughly in line with previous
 estimates from yeast and human systems. In addition to the experimental data the authors attempt to
 address the question how prevalent both transport mechanisms are. They estimate that only some 10%
 have ID linkers and NLSs.
 This paper is in my opinion an important and novel paper that would be very suitable for publication in
 nature communications after a few controls and textual changes are considered.
 Main concerns:
 1) the dual-channel co-tracking SPEED microscopy of the double tagged LBR is important since it
 shows that single molecules of LBR pass the NPC as a full-length protein and membrane embedded.
 Similar experiments are not included for Lap2β. This is a potential problem in the interpretation of the
 data for Lap2β, as, obviously, the presence of truncated versions encoding only the nucleoplasmic,
 soluble domain of LBR could easily explain the signal the central channel. I realize that doing dual
 channel co-tracking of Lap2β would involve a very significant extra effort and this is not required in
 my opinion. I would however like to see western blot and microscopy images of the cells expressing
 the lagged Lap2β, so that readers can be convinced that the data very likely reflects full length
 proteins and not a degradation product of Lap2β.
 2) The estimate that ~9% of the inner nuclear membrane proteins may encode an ID domain and an
 NLS is really only an estimate as should be referred to as such (also in the abstract). The assumption
 that all these “extend intrinsically disordered (ID) domains containing nuclear localization signals
 (NLSs) into the central channel for directed nuclear transport” is also really only an assumption and
 should be referred to as such. To clarify the analysis done, I have three more suggestions: Please
 provide the list of 46 INM and 11 ONM proteins that were included in the analysis. Line 190 (methods)
 Please indicate the rationale for taking SeqNLS scores higher than 0.86 as ‘sufficiently strong’ NLSs.
 Please elaborate what “made sense in a biological manner for facilitated transport through the center
 of the NPC “ means.
 Minor comments
 3) I have few suggestions towards placing the work in the context of the literature.
 a. The two papers that were published in 2015, by the Ellenberg and by the Kutay lab (Boni et al, JCB
 2015, Ungricht et al JCB 2015) should be discussed. Both groups used life imaging and computational
 
techniques to show that a diffusion-retention mechanism can explain the import of INM proteins. In
 both papers Lap2β and LBR are studied, like in this manuscript. The estimates of transport kinetics in
 Boni et al match nicely the ones reported here (5.6 molecules per minute per NPC versus 4.6
 molecules/NPC/min), but while the numbers match, the conclusion of Boni et al is that their data
 supports a diffusion retention model for INM protein transport.
 b. In the introduction Line 107-109 is an incorrect statement: In Meinema et al (2011) the
 endogenous ID region of the yeast proteins was used and in Kralt et al. MolBiol Cell 2015 the full
 length and endogenously expressed protein (native promotor, native chromosomal location) was used
 to study the NLS. If the authors meant to say that that thus far there were no papers that studied the
 relevance of an ID linker in the context of the full length and natively expressed protein, that would be
 correct.
 c. In the Discussion: Line 341-343. The experiments do not address this concern directly (different
 proteins, different cells). Laba et al (Cells, 2015) confirms the transmembrane nature of the Heh2-
 derived truncated proteins.
 3) Line 255: there is no data to support that the R74T mutation indeed results in the disruption of NTR
 binding. Is there biochemical evidence to support this statement? Or is there data to show
 mislocalisation of GFP-NLS(R74T) fusions in cells? Ma et al 2007 J Cell Sci can be cited to support that
 imp beta is bound.
 4) Other:
 Line 220-223: Lap2β and Lapβ are used both throughout the manuscript.
 Line 261: Lapβ ΔNLS: actually the NLS is not deleted but mutated: R319T and R320T. Please mention.
 Line 160 (methods) range not rage.
 Line 181 (methods) In the case of LBR it reads that the no of copies per cell was based on a previous
 estimate; what was used for the other proteins?
 Line 218-223 (methods). I do not understand which figures or experiments this related to.
 Line 265-268: this paragraph is slightly confusing. Maybe add: ...and roughly 80% of the length of the
 nucleoplasmic region still present, LBR required these ID Linker regions to transit....etc.
 Smovie 2 does not work
 Reviewer #3 (Remarks to the Author):
 This paper has studied how transmembrane proteins in their inner nuclear membrane are transported
 to the inner nuclear membrane using live cell high-speed super-resolution single-molecule microscopy.
 Authors found that either just the peripheral channels or both central and peripheral channels together
 are used for the transport depending on their nucleoplasmic signals. They succeeded in visualizing
 pathways and single-molecule trajectories going through the central and peripheral channels
 distinguishing the positions of the central and peripheral channels and pore membrane lumen.
 However, the paper has substantive issues in description, which makes the paper hard to comprehend.
 Major:
 1. There is a problem with the logical description. In addition, some paragraphs are too long in the
 Results and Introduction sections. The Discussion section has insufficient contents.
 2. Abstract is inappropriate as summary. It does not reflect the entire contents, and combining
 Abstract and Significance Statement makes a better summary. Furthermore, for example, the value of
 “~9%” is described only once in the Results section, and Fig. 4E is referred. However, Figure 4E and
 its legend has no description of the clear basis for calculation, and the accuracy of the numerical value
 of 9 is not described.
 3. The title is not suitable. The word “Nucleoplasmic signals” of the title subject is used only once in
 
the Significance Statement section through the whole manuscript, and no explanation is described
 even though the word is not in common use. The word “simultaneously” is also used, but it is not clear
 with which nucleoplasmic signals promote simultaneously.
 4. It is written that the route precision is ~2 nm at line 164 in the Result section, and 0.90 ~ 1.42 in
Table S2 based on the simulations (Figs. S8-10). However, the validity of using the values is not clear.
It is considered appropriate to use the standard deviation of the distribution or that of data from
independent measurements (not standard error of the mean SEM; more precisely, Student's t value
multiplied by SEM) as the precision and error of the route and diameter.
Minor:
 1. The sentence “~9% additionally extend ...” in Abstract is unclear, since the subject of the sentence
 is not clear.