The authors analyze the role of Cereblon in NK functions focusing on its potential involvement in actin cytoskeleton reorganization during the formation of the immunological synapse between NK and target cells and during NK polarization in response to chemokines. They unveil Rac1 as a functional target of Cereblon.

Then authors first analyze the intracellular localization of Cereblon by biochemical approaches (subcellular fractionation and western blot) and immunofluorescence and confocal microscopy.

From microscopy images in Fig 1 the authors conclude that Cereblon concomitantly relocalizes with F-actin to the immunological synapse of NK-target cells and the front edge of chemokine treated NK cells.
In my opinion microcopy evidences a too poor to support these conclusions. Cereblon immunofluorescence shows staining all over the cell, including the cell cortex, but not particularly enriched in areas with more F-actin intensity in isolated NK cells (Fig 1 B top). Moreover, the first image of an immunological synapse (Fig 1 B, bottom) shows Cereblon overlapping with F-actin, in a target cell displaying intense Cereblon staining. This makes difficult to know from which cell the Cereblon staining is coming.
These worries are further supported by Fig 1 C, top, in which the authors show a grape of what it looks two target (big cells) and two NK (small cells). What it is the most striking there is the accumulation of Cereblon at the junction between the two big target cells with much more faint staining at the junctions with NK cells. Therefore, to this reviewer, no sufficient evidence is provided here for relocalization of Cereblon to the immunological synapse within the NK cell.
Not obvious to me either the colocalization with F-actin. Although the overlap in Fig 1 B bottom appears clearer, it is much less obvious in Fig 1 C bottom, in which the two patterns of fluorescence do not really follow each other.
In Fig 1 D that authors analyze NK cells polarized in response of chemokines. They conclude that Cereblon localized at the lamellipodial leading edge where actin polymerization occurred. I still do not fully agree with the conclusion. What I observe there, which may be also very interesting is a Cereblon+ intracellular vesicular-like compartment that polarizes just behind the F-actin rich leading edge. The image is quite noisy and the leading edge looks very large lacking texture. Again, F-actin and Cereblon staining although overlapping in part do not have the same morphology.

In my oppinion, Fig 1 indicates a concomitant polarization of Cereblon and F-actin in close subcellular areas but I would not call it colocalization, but just partial overlap in both immune synapses and chemokine polarized cells. This is also evident in subsequent figures performed with NK92 cells as Sup Fig 1 A, which shows synapses with more accurate structure.

Therefore, the authors need more convincing and resolutive images of synapses able to solve whether Cereblon relocalization occurs in NK cells or it is just an effect of cell junction that may occur between NK and target cells as with two target cells. A choice of target cells expressing low levels of Cereblon could help on this. Moreover, get more resolutive images that facilitate enlargement to appreciate the texture between Cerblon subcellular structures and F-actin could help to solve whether Cereblon is linked to F-actin or just concomitantly polarizes to the synapse and to the proximity of the leading edge. The later possibility would not invalidate the authors’ hypothesis of the involvement of Cereblon in NK migration and function.
Adding to Fig 1, Sup Fig 1 A panel would actually improve this figure since it is of much better quality. Zooming the synapse region would also help.

The rest of functional assays described in the other figures appear to me robust and convincing.
The link the authors find between Cereblon and Rac1 activation in target cell or chemokine) stimulated cells may deserve some additional experiments to analyze the involvement of Cereblon in actin cytoskeleton remodeling in both NK synapses and chemokine stimulated polarized T cells. This would improve the message the authors wish to convey with the current Fig 1.

Minor points
To facilitate reading and image interpretation, the authors should label the NK and the target cells on the images and add some arrows pointing the synapse events thy are referring to.

Indicate in the figure panels when NK primary or NK92 tumor cells are being used would help faster reading and interpretation of figures.

The authors should take advantage of their ability to inhibit Cereblon expression with shRNA vectors to test the specificity of the anti-Cereblon Ab in immunofluorescence analysis.

The authors responded to my queries with higher quality new data and adapted the text. I think the manuscript is now fine for publication in EJI