Actin’s evolving roles: A multi-billion-year-old protein learns new tricks

From the Malik Lab, Basic Sciences Division

If you cannot get rid of the family skeleton, you may as well make it dance. -George Bernard Shaw

The skeleton. It’s a strong structure, and a stronger symbol, though one rife with contradiction. A symbol of frailty and decrepitude, when in truth it gives our bodies strength. A symbol of regret, when in truth it serves as the base, quite literally, of life’s most joyous activities. A symbol of death, when in truth it is a living and breathing thing that plays host to a vibrant community of cells. In fact, to my mind the skeleton serves, ironically, as among the most powerful symbols of the continuity of life. Though we look quite different on the outside, compare the skeletons of a human, a kangaroo, a shark, a bird, and you can’t help but see how alike we all are – a testament to our common origins and to the largely unchanged nature of this ancient structure.

Within our cells exists an analogous, and even more ancient, structure – the cytoskeleton. Composed of strong protein filaments, the cytoskeleton provides physical support to cells and helps generate the mechanical forces required for behaviors like migration and division. But this structure, too, contains an apparent contradiction. Actin, a core cytoskeletal protein, and a group of related proteins known collectively as the actin-related proteins (Arps) evolved a few billion years ago, give or take, and have remained largely unchanged over the intervening eons. “Because of its interactions and functional importance, actin evolves under stringent evolutionary constraints. For example, despite being separated by 800 million years of evolution, actin proteins from Drosophila melanogaster and Homo sapiens are 98% identical” write Drs. Courtney Schroeder and Harmit Malik, members of the Fred Hutch Basic Sciences Division, in a newly published paper in eLife. Despite this extreme conservation, Drs. Schroeder and Malik made the surprising discovery that one Arp in Drosophila melanogaster has rapidly evolved to take on important new roles in the animal’s development.

“[Ancient] canonical Arps significantly diverged from each other early in eukaryotic evolution, but now evolve under stringent evolutionary constraints. Many eukaryotic genomes also encode evolutionarily young, rapidly evolving “non-canonical’ Arps,” the authors explain. Interestingly, these non-canonical Arps appear to be expressed exclusively in the male germline, indicating that they probably play some role in male fertility, although their functions are mysterious. To better understand how young Arps evolved to integrate into the very old actin machinery, the authors examined one such gene: Arp53D. They first compared this gene between species to date its appearance to around 65 million years ago – recently enough to be considered young but long ago enough that its continued existence suggest it likely has an important function (“deleterious or non-functional genes are…lost within a few million years in Drosophila genomes,” they point out). Further, the gene’s recent mutational signature shows that it’s been evolving rapidly, again indicative of a functional role. They next examined what role this gene may be playing in the testes, and found that it localizes to two actin-based structures with important roles in sperm development: the fusome, a structure associated with germ cell division, and the actin cone, a structure involved in the formation of individual sperm. Surprisingly, though, they found that knocking out Arp53D did not impair male fertility; in fact, the males lacking this gene were more fertile, in stark contradiction to its evolutionary history. “Our findings thus still leave unanswered the question of why Arp53D was largely retained over 65 million years of Drosophila evolution,” they concluded.

Dr. Malik is no stranger to such contradictions. In his experience, if an evolutionarily retained gene appears to have negative consequences in one context, those are likely balanced by positive consequences in another context. Indeed, when they mixed flies with and without Arp53D and let the population evolve, the flies with Arp53D clearly won out, suggesting its presence is indeed evolutionarily advantageous. But in what context is Arp53D playing its beneficial role? “Although Arp53D is most abundantly expressed in adult testes, there is also weak expression in other tissues and developmental stages,” they note. They found that loss of Arp53D reduced the fertility of females under heat stress, as well as impaired development of the early embryo. This young actin-associated gene has, then, evolved to play an important role in Drosophila, although not in the way that Drs. Schroeder and Malik had expected.

“We were surprised to find that [Arp53D] plays important roles beyond the testis. Not only does this suggest that other ‘testis’ Arps found in other species may play non-male roles, but it also serves as a warning to not use enrichment of expression as a sole guide for studying function,” said Dr. Schroeder. She is also excited about the implications of this work to understand Arp function in a wide range of organisms. “It may seem esoteric to study a divergent, ‘testis’ Arp found only in fly species, but there are actually many divergent Arps that are enriched in the testis across phyla—from flies to humans. And we have no idea what they do! Arp53D served as a great test case for probing a testis Arp’s function genetically…what are other testis Arps doing across species? I think there is most likely a functional commonality shared among all ‘testis’ Arps, and evolution has selected for divergent Arps multiple times for roles in stress and development.” Dr. Schroeder has just begun a faculty position at the University of Texas Southwestern Medical Center, where she will continue to unlock the mysteries of Arp evolution and function.

arp53d in actin cones
Localization of Arp53D to actin cones in the fly testes. Image provided by Dr. Courtney Schroeder

This work was supported by the National Institutes of Health, the Jane Coffin Childs Memorial Fund for Medical Research, and the Howard Hughes Medical Institute

Fred Hutch/UW Cancer Consortium member Harmit Malik contributed to this work

Schroeder CM, Tomlin SA, Mejia Natividad I, Valenzuela JR, Young JM, Malik HS. (2021) An actin-related protein that is most highly expressed in Drosophila testes is critical for embryonic development. Elife. 10:e71279.