The ins and outs of calcium during wound healing

Wound repair is an essential component of life. Without it any minor cuts or scrapes could be extremely damaging or even lethal. Paper cuts are bad enough already without having to consider them becoming life threatening. There are many components of the wound repair response, but one of the earliest signals is the influx of calcium into the damaged cells from the extracellular space. Calcium is extremely important in many aspects of cell biology.  During the wound response it activates downstream molecules which are integral for membrane closure and repair. Annexins (Anxs) are calcium-dependent molecules which are rapidly recruited to sites of cell membrane injury and aid in the repair process. There are many forms of Anxs, with names differing in alphanumeric suffixes, such as AnxA1 or AnxB11, all with slightly diferent yet often overlapping contributions to the cellular response. Anxs contribute to repair by modulating the cytoskeleton, the dynamic network of proteins that includes actin which provides a cell with its shape and allows it to move. Characterizing the roles of individual Anxs and how they respond to calcium influx during wound repair was the main goal of Dr. Mitsutoshi Nakamura, a postdoc in the Parkhurst lab from the Basic Sciences division, in their recent preprint in BioRxiv.

The research team used Drosophila, the common fruit fly, as a model of cellular wound repair to generate beautiful fluorescent images of Anxs and actin surrounding wounds made by laser ablation. Previous work by the Parkhurst lab had uncovered the role of AnxB9 in recruiting RhoGEF2, which has functions in actin rearrangement. Drosophila have only two other Anx, AnxB10 and AnxB11, that regulate two other RhoGEF proteins, RhoGEF3 and Pbl, which are still recruited when AnxB9 was knocked down. This previous result “led me to investigate whether the other two Anxs regulate the other RhoGEFs during cell wound repair”, explained Dr. Nakamura.  To address this, they generated small wounds in embryos of transgenic flies which expressed fluorescently labelled AnxB9, AnxB10 and AnxB11 and observed the immediate dynamics of these proteins to the wound site. With impressive speed, the Anx proteins were all recruited to the site of injury in less than three seconds but did display differences in their arrangement once they’d arrived. Visible in the images is an actin ring and actin halo, two patterns of actin formation that are typical of this model, as well as patterns of the Anx proteins overlayed. The team observed that AnxB9 formed most visibly well inside the wound edges, surrounded by AnxB10, followed by a more dispersed arrangement of AnxB11 (see image). Considering how fast the recruitment response occurred, Dr. Nakamura highlighted that “it is surprising that a cell could generate such patterns so quickly upon wounding”. Next, to characterize the role of the different Anxs in the wound repair process, they observed that knockdown of any of three Anxs resulted in wound overexpansion and lower actin recruitment, but knockdown of AnxB9 or AnxB10 resulted in additional responses such as slower wound repair and decreased actin ring width. Together, these initial results indicated overlapping and distinct functions of these three Anxs in wound repair.

To then investigate the roles of AnxB10 and AnxB11 in recruiting RhoGEFs, the team employed additional fluorescently tagged Drosophila models for RhoGEF3 or Pbl. Interestingly, they observed that RhoGEF3 recruitment was lost, but that Pbl recruitment was retained, in both AnxB10 and AnxB11 knockdown backgrounds. Loss of RhoGEF3 recruitment was partially returned when the researchers added an actin stabilizing compound, indicating to them that the Anxs likely work together to recruit RhoGEF3 through actin dynamics. In their previous work, they had found that the recruitment of RhoGEF2 was also dependent on actin stabilization, and so they additionally asked what impact AnxB10 and AnxB11 may have on this RhoGEF. Again, they found that in either knockdown model, RhoGEF2 recruitment was lost. However, perhaps more intriguingly, actin stabilization was not able to rescue this response, pointing to additional roles of these Anxs on actin dynamics in addition to stabilization.

Since Anxs are calcium-dependent proteins, the researchers wanted to address how extra- and intra- cellular calcium may impact Anx recruitment. A particular feature of Drosophila embryos which helped facilitate asking this question is that they are surrounded by an impermeable ‘wall’ called the vitelline membrane. Taking advantage of this, they injected a calcium chelator into the space between the vitelline membrane and the embryo or directly into the embryo, effectively depleting the extra- or intra-cellular calcium, respectively. Following the loss of extracellular calcium, none of the Anxs were recruited to the wound site. However, loss of intracellular calcium still resulted in recruitment of Anxs but in abnormal patterns. Even more interestingly, when extracellular calcium was only moderately depleted, this resulted in abnormal Anx organizational patterns similar to loss of intracellular calcium. Together, this highlighted to the research team that the quantity of the calcium influx was very important to Anx recruitment during wound responses. In line with their observations about loss of Anxs on wound repair responses, loss of extracellular calcium resulted in an inability for the wound to close. However, only with loss of intracellular or Ex-half calcium did wound exhibit over expansion and delayed closure, with Ex-half exhibiting slower responses between the two.

All together the research team concluded that Drosophila cells can sense the source and quantity of calcium at the moment of injury, and that this source provides very valuable information for how the cells recruit Anxs and subsequently RhoGEFs to the wound site. Building off their previous study of AnxB9, here they further built the picture on how the recruitment and organization of the three Drosophila Anx and RhoGEF proteins are indispensable for wound repair. Since calcium is a vital secondary messenger in many aspects of biological functions, understanding the mechanism through which it regulates wound responses could inform a variety of pathologies, including muscular dystrophy and diabetes, which have altered intracellular calcium levels and impaired wound repair. Dr. Nakamura describes the previously considered role of calcium in wound responses as a “kick-starter” but was intrigued to see his data support a more nuanced contribution.

The spotlighted research was funded by the National Institutes of Health and an endowed chair from Fred Hutchinson Cancer Center.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Susan Parkhurst contributed to this work.

Mitsutoshi Nakamura, Susan M. Parkhurst. Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds. bioRxiv 2023.12.03.569799; doi: https://doi.org/10.1101/2023.12.03.569799

Nakamura M, Verboon JM, Parkhurst SM. Prepatterning by RhoGEFs governs Rho GTPase spatiotemporal dynamics during wound repair. J Cell Biol. 2017 Dec 4;216(12):3959-3969. doi: 10.1083/jcb.201704145. Epub 2017 Sep 18. PMID: 28923977; PMCID: PMC5716286.