PREreview of Microtubule-dependent cell polarity regulates skin-resident macrophage phagocytosis and directed cell migration

The manuscript by Peterman et al. investigates the role of microtubule dynamics in Langerhans cell morphology, phagocytosis, and directed migration in the epidermis. Through live imaging in zebrafish explants, the study shows that microtubules originating from a perinuclear microtubule organizing center (MTOC) guide the extension of dendrites for effective debris engulfment and enable precise migration toward tissue damage. When microtubules are disrupted, Langerhans cells become less efficient at phagocytosis and lose directional control during migration. These defects are linked to altered actin cytoskeleton polarity through the RhoA/Rho-associated kinase (ROCK) signaling pathway. The findings highlight how microtubule-dependent cell polarity enables immune cells to respond effectively within complex epithelial microenvironments. We found this study to be well-written and containing high-quality data that advances the fields of microtubule and immune cell biology. Overall, the data presented in this manuscript are done well and support the claims made by the authors. We outline some major and minor adjustments aimed at aiding the clarity of reporting and presentation.

Major comments

Page 10, Lines 286-289: We felt it was somewhat unsupported that F-actin accumulation in the trailing half of the cell was “consistent with the idea depolymerizing microtubules increases RhoA activity at the rear of the cell.” While the data clearly show a disruption in F-actin distribution with nocodazole treatment, we felt it was not clear that this would increase F-actin in the trailing half rather than evenly throughout the cell. Our lack of expertise in the field may lead to our misinterpretation of this sentence, however we felt additional explanation is needed (e.g. on the Lifeact-mRuby reporter) to clarify the section and support the conclusions drawn. Consider including a schematic of the model to ease interpretation of the data shown in Figure 4.

Minor comments

Page 2: It may be more effective to explicitly introduce RhoA/ROCK in the introduction rather than first mentioning it on page 10. This could connect your ideas more thoroughly, even if it’s just a brief mention in the introduction.

Page 3, Line 102: You mention that the mpeg1.1 promoter labels multiple macrophage populations. Is there a concern that you’re labeling more than Langerhans cells in the epidermis, and that cells could be confused due to their altered morphology during the treatment?

Page 3: The writing may be clearer if all acronyms (i.e. EMTB as ensconsin microtubule binding domain, EB3 as end-binding 3) are defined at their first use.

Figure 1D: We found this panel somewhat difficult to interpret. Consider showing this panel in two dimensions displaying the percentage of EMTB+ dendrites as a function of the number of dendrites per cell.

Figure 1K: It appears that the nocodazole treatment has one outlier (value of 100 µm). Does removing this datapoint change the significance of the treatment on maximum dendrite length?

Figure 2E: It was unclear how the distance between the MTOC and phagosome was determined, i.e. whether the phagosome was measured from the point most distal or proximal to the cell body.

Figure 2J: We thought your data would be most effective if you showed both the number and percentage of engulfment events for both control and nocodazole-treated cells to demonstrate how many events happened under each condition.

Figure 3B: It appears that there are fewer Langerhans cells present in nocodazole-treated samples. Is this a significant impact or just coincidence in the images shown? Furthermore, could off-target effects or toxicity be impacting the migration differences seen here?

Page 10, Line 263-264: There may be a typo here, where it‘s omitted that the “Langerhans cells had a smaller meandering index” were nocodazole-treated.

Figure 4: A quantification of RhoA activation, e.g., using immunoblot, would be stronger evidence to support the conclusion that disruption of microtubules alters actin polarity through the RhoA/ROCK signaling pathway. This may be technically challenging: can one compare pulled-down microtubules to quantify RhoA binding between treated and non-treated?

Figure 5: We’re interested to see if nocodazole-treated Langerhans cells would respond similarly to vehicle-treated (5C) or paclitaxel-treated (5D-E), especially considering the impacts of nocodazole on dendrite morphology (decreased cell dendrite number with increased length) you showed in Fig. 1G and Supplemental Video 3. We don't think this is a necessary experiment but may be worth including to provide alternative evidence of the impact of microtubule alteration on cell migration. We also found the placement of Figure 4 to disrupt the line of thinking connecting Figure 3 and 5. Consider moving Figure 5 after Figure 3 for logical flow, as Figure 4 is more mechanistic and addressing the question of the role actin plays in this process.

Page 14, Line 385: It seems there may be a typo here, where “n=128 cells counted from N=13 scales” should include that these are in paclitaxel conditions.

Page 14, Line 395: You mention that “acute chemical perturbations” were used in this paper. We thought that the laser ablation and/or scratch injury assays may be more accurately described as a physical or mechanical perturbation rather than chemical, but this may be from a lack of familiarity with writing conventions within the field.

Page 15: In the second paragraph under “Cell motility,” there’s no name given for the image processing software used, which we think it would be helpful to include.

Methods, Line 522: Could you write the exact percentage of DMSO used for the vehicle controls either here or directly in the figure legends.

Supplemental Videos: We found your supplemental videos extremely informative. Would it be possible to include these in the main text?

Madison McReynolds and Mandkhai Molomjamts (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Sally Abulaila, Kim Kissoon, Michael Kwakye, Madaline McPherson, Habib Ogunyemi, Octavio Origel, and Warren Wilson.

Competing interests

The authors declare that they have no competing interests.