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Fish basal epidermal cells, known as keratocytes, are well‐suited for cell migration studies. In vitro, isolated keratocytes adopt a stereotyped shape with a large fan‐shaped lamellipodium and a nearly spherical cell body. However, in their native in vivo environment, these cells adopt a significantly different shape during their rapid migration toward wounds. Within the epidermis, keratocytes experience two‐dimensional (2D) confinement between the outer epidermal cell layer and the basement membrane; these two deformable surfaces constrain keratocyte cell bodies to be flatter in vivo than in isolation. In vivo keratocytes also exhibit a relative elongation of the front‐to‐back axis and substantially more lamellipodial ruffling, as compared to isolated cells. We have explored the effects of 2D confinement, separated from other in vivo environmental cues, by overlaying isolated cells with an agarose hydrogel with occasional spacers, or with a ceiling made of polydimethylsiloxane (PDMS) elastomer. Under these conditions, isolated keratocytes more closely resemble the in vivo migratory shape phenotype, displaying a flatter apical‐basal axis and a longer front‐to‐back axis than unconfined keratocytes. We propose that 2D confinement contributes to multiple dimensions of in vivo keratocyte shape determination. Further analysis demonstrates that confinement causes a synchronous 20% decrease in both cell speed and volume. Interestingly, we were able to replicate the 20% decrease in speed using a sorbitol hypertonic shock to shrink the cell volume, which did not affect other aspects of cell shape. Collectively, our results suggest that environmentally imposed changes in cell volume may influence cell migration speed, potentially by perturbing physical properties of the cytoplasm.

Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil‐like HL‐60 cell lines expressing GFP‐β‐actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL‐60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal β‐actin. Surprisingly, we recovered viable monoallelic GFP‐β‐actin cells as well as biallelic edited GFP‐β‐actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP‐β‐actin incorporates into the F‐actin network in biallelic edited cells at similar efficiencies as normal β‐actin in unedited cells. Additionally, the edited cells significantly upregulate γ‐actin, perhaps helping to compensate for the loss of normal β‐actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well‐suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.