On 2022-04-13 23:23:11, user Nathan Ewing-Crystal wrote:
Naive T cell migration between and within lymphoid organs is essential for detecting cognate antigen on antigen presenting cells; this migration is largely driven by CCL21-CCR7 signaling. This manuscript explores the WNK1 pathway, which is induced by CCR7 signaling and results in ion influx necessary for T cell migration (as shown by this group’s previous work). The authors analyze each WNK1 pathway member’s contribution to migration speed and cell volume through selective ablation and inhibition of proteins within the pathway. They further uncover a spatial enrichment of these proteins at the leading edge of migrating T-cells; localized water entry at this leading edge results in localized swelling and increasing membrane-F-actin spacing. They propose that WNK1 signaling facilitates F-actin polymerization via a Brownian ratchet mechanism.
The major success of this paper is in developing a concise model for the role of WNK1 pathway activation and subsequent ion influx in cell migration through localized water entry facilitating F-actin polymerization. Moreover, the authors decisively link chemokine signaling not only to directional decision-making but also to the physical act of cell migration.
Only minor aspects of the model require clarification; specifically, it is unclear to what extent WNK1 signaling drives directional migration in addition to physical migration (see below). Simple experiments (or reanalysis of existing ones) might be sufficient either to clarify the model or to demonstrate that the uncertainties in question will require extensive further investigation.
Overall, this paper deepens the field’s molecular understanding of chemokine signaling, specifically the extent to which intracellular structural reorganization is a necessary component of chemokine-induced migration.
Major Points:<br />
-While the data show a WNK1-dependent migration speed effect, they also suggest that this may not be the only effect of WNK1 on cell migration. If WNK inhibitors reduce T-cell shape polarization and increase circularity (supplemental Fig. 1D), is it possible that WNK regulates directional movement in addition to migration speed? Alternatively, given that migration speed is determined from timelapse images multiple seconds apart, is it possible that the measured difference in speed is actually detecting an impaired ability to maintain directional movement? An analysis of displacement vs. distance over the imaging timecourse would provide insight as to whether WNK-inhibited T-cells also display directional dysfunction. Alternatively, providing CCL21 as gradient rather than as a homogenous stimulus could enable an analysis of whether WNK signaling is necessary for accurate directional movement.
-The claim that “WNK1 pathway proteins and their activities accumulate at the leading edge of the cell” raises a few unresolved questions. Specifically, does a pre-designated leading edge accumulate WNK1/OSXR1/other WNK signaling effectors? Or do local accumulations of these proteins determine the leading edge of the cell? While a textual revision of this claim could reduce the imprecision, the question raised is interesting and worth answering. For example, when migrating cells switch direction (either spontaneously or in response to an experimentally altered chemokine gradient), do WNK1 and effector proteins begin to accumulate on the new leading edge prior to directional switch, or afterwards? If the former is the case, implying that WNK1 accumulation and signaling may drive selection of a leading edge, WNK1 may play a fundamental role not only in chemokine-driven physical migration but also in the response to chemokine gradients. If the latter is the case, implying that a leading edge recruits WNK1, this signaling pathway would instead drive migration in whatever direction has already been selected. Either way, additional analysis or context would clarify the role of WNK1 signaling in the overall response to chemokine signaling and the extent to which WNK1 drives “movement” vs. “directional migration.”
Minor Points:
-To the reader unfamiliar with in vitro migration assays, the rationale for the inclusion of ICAM - a signaling molecule that could interfere with analysis of chemokine signaling pathways - is unclear; consider providing a textual explanation for this decision.
-As stated, OXSR1 and STK39 both phosphorylate SLC12A2; is their role redundant, are both necessary? (Specifically, if the experiments in Fig 1B are repeated with OXSR1-/-/STK39+/+ and OXSR1+/+/STK39T243A/T234A, would we expect to see the same decrease in migration speed as OXSR1-/-/STK39T243A/T234A, no loss in migration speed, or an intermediate phenotype?)
-If AQP3 is the main water channel expressed on CD4 T cells, why does inhibition of this channel not change basal volume of T-cells (as with Wnk1/Osxr1 inhibition)? Does this data suggest a functional redundancy of water channels, as is suggested with ion channels?
-Consider providing an explanation or reference for the selection of CDC42/CD44 as definitive markers of the leading and trailing edge.
-In Figures 2A-C, it appears that WNK1 and downstream effectors are somewhat enriched at the trailing edge of cells relative to the middle (though to a lesser extent than at the leading edge); speculation as to the significance and cause of this enrichment would help clarify the data. Additionally, the cell expressing GFP alone appears to have a shorter distance from leading to trailing edge, which may also contribute to this effect.
-In Figure 3, plasma membrane (PM) and actin signals generated via instant structured illumination microscopy are normalized, and distances between peaks are measured. These peaks are further away at the cell’s leading edge than on the sides, leading to the interpretation that there is more distance between actin and membrane at the leading edge. However, this interpretation ignores the possibility of a thicker and/or differently distributed actin layer at the leading edge, with the densest region (corresponding to the “peak” of the normalized signal) further away from the membrane simply because the layer of actin is thicker. This scenario would produce identical normalized data despite involving no difference in the distance between the edge of the actin layer and the edge of the membrane. While figure 4 suggests that there is less membrane-proximal actin at the leading edge, the actual distance between membrane and the actin that is present remains unclear. Consider providing un-normalized data to help clarify this interpretation (with similar peak intensities between leading and non-leading edges implying similar actin layer distributions). Alternatively, consider comparing the strength of the (non-normalized) actin signal at the location of the membrane signal across cell membrane locations.
-In Figure 3B, the actin and membrane signals appear to be normalized to peak actin and peak membrane fluorescence, respectively. In contrast, in Figure 3C, both signals appear to be normalized to peak membrane fluorescence. Consider clarifying whether this is the case, and why these distinct strategies are utilized.
-Could the negative effect of hypotonic solutions on the migration speed of phenotypically normal T-cells arise from water entry through non-AQP3 water channels? (Presumably any additional entry of water through AQP3, concentrated at the leading edge, would further increase migration speed). If this is the case, wouldn’t non-specific cell-wide water entry, and subsequent diminishing of directional migration, still occur in WNK-inhibited T-cells? The use of superresolution imaging and/or membrane-proximal actin visualization in experiments involving altered tonicity could provide insight as to where hypotonicity-induced water entry is occurring. Additionally, speculation as to why the benefit of bypassing a blocked WNK pathway overcomes the deficit caused by non-leading-edge-specific water entry (if visualized) would be helpful.
-Could labeled materials (e.g. water and sodium isotopes) or depletion of individual ions (i.e. sodium, potassium, etc., one at a time) be used to further characterize the influx of ions and water proposed in this manuscript? While possibly outside of the scope of this paper, consider discussing this (or other biochemical methods) as part of a set of proposed future experiments.
Stylistic notes:<br />
-The order of the data as presented in the figures does not match the order of data as presented in the text. For example, the first half of figure 1 is discussed, followed by the entirety of figure 2, followed by the second half of figure 1. Consider revising the text to reflect the order of the data in the figures, or vice versa.
-Consider including figure 4k as a graphical abstract for the published paper (for people unfamiliar with the signaling pathway and who get confused by the acronyms). Further, on figure 4k, consider adding a visual depiction of G-actin monomer addition to the F-actin filament tips following the depiction of the membrane swelling.
-Consider stating the full names of proteins discussed before using their respective acronyms (e.g. lysine deficient protein kinase 1 for WNK1), to help readers unfamiliar with these proteins.
Note: We cannot offer expert feedback on the superresolution microscopy methods used, other than the already raised questions about the interpretation of the resulting data.
Nathan Ewing-Crystal (nathan.ewing-crystal@ucsf.edu)<br />
Catherine Kuhn (catherine.kuhn@ucsf.edu)