ActuAtor, a molecular tool for generating force in living cells: Controlled deformation of intracellular structures

Abstract

SummaryMechanical force underlies fundamental cell functions such as division, migration and differentiation. While physical probes and devices revealed cellular mechano-responses, how force is translated inside cells to exert output functions remains largely unknown, due to the limited techniques to manipulate force intracellularly. By engineering an ActA protein, an actin nucleation promoting factor derived from Listeria monocytogenes, and implementing this in protein dimerization paradigms, we developed a molecular tool termed ActuAtor, with which actin polymerization can be triggered at intended subcellular locations to generate constrictive force in a rapidly inducible manner. The ActuAtor operation led to striking deformation of target intracellular structures including mitochondria, Golgi apparatus, nucleus, and non-membrane-bound RNA granules. Based on functional analysis before and after organelle deformation, we found the form-function relationship of mitochondria to be generally marginal. The modular design and genetically-encoded nature enable wide applications of ActuAtor for studies of intracellular mechanobiology processes.

Drops and fibers — how biomolecular condensates and cytoskeletal filaments influence each other

“…for centrosomes during mitotic exit [72]. To further investigate how condensates are deformed by internal forces, a novel tool called 'ActuAtor' can help, which triggers localized actin polymerization in cells in response to chemical or optical stimuli [97]. Actin localized to stress granule proteins polymerizes within these stress granules and disperses the anchor proteins of the 'ActuAtor'.…”

Disentangling the biological information encoded in disordered mitochondrial morphology through its rapid elicitation by iCMM

“…1b) to produce the functional iCMM effector. Similar to ActuAtor, a codon-optimized ActA functionally homologous to mActZ-FY 24 , cytosolic mActZ-FY recruitment to mitochondria by rapamycin treatment obviously transformed mitochondrial morphology to small, dot-like structures of almost equal size, in cells expressing mActZ-FY and Tom20-CR (Fig. 1e, h, and k, Supplementary movie 4).…”

“…1b). Although YF has been used as a control effector in various experimental settings 23,24 , we found that once the diffusive YF was substantially translocated to the outer surfaces of mitochondria, which was circumscribed by anchor proteins—which consisted of mitochondrial targeting sequences derived from Tom20, enhanced cyan fluorescent protein (ECFP), and FRB (termed Tom20-CR)—mitochondrial morphology changed from a basal reticular thread-like structure to large punctate structures (Fig. 1c, Supplementary movie 1).…”

“…Lastly, we optimized a previously designed interspecies fusion protein consisting of aa 30–262 of Listeria monocytogenes Actin assembly-inducing protein (ActA), codon-optimized for usage in mammalian cells 24 , and aa 2–380 of human zyxin (termed mActZ), and being structurally and functionally similar to full-length ActA 28 . As ActA converts actin polymerization into a motile force 29 , we hypothesized that the mechanical force generated by actin polymerization could alter mitochondrial morphology.…”

“…The mActZ-FY effector was produced by subcloning sequences coding aa 30–262 of codon-optimized Listeria monocytogenes serovar 1/2a ActA (UniprotKB-P33379) and aa 2–380 of human zyxin (UniprotKB-Q15942) into an FY vector containing the FKBP F100Y mutant and EYFP, at the N-terminus between the Xho I and EcoR I sites. The ActA nucleic acid sequence was optimized for mammalian cells 24 . The Tom20-CR anchor was produced by inserting a stop codon encoding Tom20-CR 56 after the Xho I site in the vector, using the Q5 Site-Directed Mutagenesis Kit.…”