The Natural Material Evolution and Stage-wise Assembly of Silk Along the Silk Gland

Silk fibers, with their highly ordered structure and mechanically superb properties, are produced in arthropod glands at minimal energy input and ambient conditions, a remarkable feat yet to be achieved synthetically. Due to the high instability and shear sensitivity of the silk protein feedstock, understanding silk fiber formation has been largely limited to in-vitro studies of certain gland sections, offering only a fragmented view of this process. Here, we monitor the whole silk feedstock processing in-situ, at the nano- to micron-scales, through imaging its progressive macromolecular assemblies and phase transitions along the entire Bombyx mori silkworm silk gland. This is done by combining state-of-the-art microscopy techniques, such as cryogenic sample preparation, fixation, and imaging. Our work reveals that fibroin assembles into micron-sized spherical storage "compartments" in the posterior and middle gland sections, a state that ensures its stability and avoids premature fibrillation. These compartments undergo several structural transformations along the gland and eventually disassemble at the entry to the anterior section, before the silk feedstock spinning begins. The spinning itself commences via a series of structural transitions, from the alignment of protein chains in liquid feedstock, through the formation of several fibrillated nano-structures and, in the final stage, a network of cross-linked nano-bundles, which determines the structure and properties of the final microfiber. Importantly, the length of the anterior section of the silk gland enables such gradual and balanced structural transitions. This direct imaging of silks natural formation process can help formulate a template for the transformation of fibrillar proteins into synthetic bio-fibers. DedicationThis work is dedicated to the memory of Dr. Eyal Shimoni, who was a valued colleague and a dear friend. Eyal was a vital part of this research and was essential in shaping its direction. He will be deeply missed for his intellect, mindfulness, creativity, and unwavering dedication to scientific development. Though he is no longer with us, his influence and spirit continue to inspire us in our scientific pursuits. May his passion for discovery and commitment to excellence live on through this work.