This study identifies structural and regulatory proteins involved in cell-shape determination in Haloferax volcanii, a model archaeon that forms rods and disks depending on growth conditions. Using a combination of iterative proteomics, genetics, and live-cell imaging, the researchers identified mutants that only form rods or disks. They compared the proteomes of these mutants with wild-type cells across growth phases to distinguish between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. The study also identified structural proteins, including an actin homolog named volactin, which plays a role in disk-shape morphogenesis. Using live-cell imaging, the researchers determined volactin's cellular localization and showed its dynamic polymerization and depolymerization. The results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains. The study highlights the importance of structural and regulatory components in cell-shape determination, including RdfA and DdfA, which are required for rod and disk formation, respectively. The findings suggest that cell-shape transitions are under explicit genetic control and may represent an evolutionary correlation between shape and lifestyle. The study also identifies additional shape-specific components, including proteins involved in signaling cascades and ABC transporters, which may be involved in transporting signals across the membrane. The results demonstrate the importance of volactin in disk-shape formation and its dynamic behavior in vivo, resembling dynamically unstable actin polymers. The study provides a comprehensive understanding of the structural and regulatory components involved in cell-shape determination in Hfx. volcanii, with potential implications for understanding the evolution of cell morphology regulation across domains.This study identifies structural and regulatory proteins involved in cell-shape determination in Haloferax volcanii, a model archaeon that forms rods and disks depending on growth conditions. Using a combination of iterative proteomics, genetics, and live-cell imaging, the researchers identified mutants that only form rods or disks. They compared the proteomes of these mutants with wild-type cells across growth phases to distinguish between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. The study also identified structural proteins, including an actin homolog named volactin, which plays a role in disk-shape morphogenesis. Using live-cell imaging, the researchers determined volactin's cellular localization and showed its dynamic polymerization and depolymerization. The results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains. The study highlights the importance of structural and regulatory components in cell-shape determination, including RdfA and DdfA, which are required for rod and disk formation, respectively. The findings suggest that cell-shape transitions are under explicit genetic control and may represent an evolutionary correlation between shape and lifestyle. The study also identifies additional shape-specific components, including proteins involved in signaling cascades and ABC transporters, which may be involved in transporting signals across the membrane. The results demonstrate the importance of volactin in disk-shape formation and its dynamic behavior in vivo, resembling dynamically unstable actin polymers. The study provides a comprehensive understanding of the structural and regulatory components involved in cell-shape determination in Hfx. volcanii, with potential implications for understanding the evolution of cell morphology regulation across domains.