Kinetics of Collagen Self-Assembly by Time-lapse Structured Illumination Microscopy

As the predominant extracellular matrix protein, collagen plays a key role in providing structural integrity and flexibility to connective tissues. These properties arise from collagen’s ability to self-assemble from a triple helical protein into large fibers with high tensile strength and elasticity. The importance of collagen self-assembly to functioning tissues is evidenced by the number of incurable connective tissue disorders associated with disrupted or defective collagen self-assembly. Despite this, key steps of the assembly process have yet to be characterized in detail. Long-standing methods of studying collagen assembly kinetics, primarily turbidity, were limited to ensemble measurements of collagen solutions. However, without real-time observation of individual fibrils, the link between these measurements and specific events in self-assembly remains tenuous. Time-lapse confocal microscopy of collagen self-assembly has previously been used to bridge this gap, but it is limited in sensitivity and resolution such that only later stages of assembly can be observed. Here, we use real time structured illumination microscopy (SIM) to observe growing fibrils from the earliest stages and characterize the kinetics of collagen self-assembly on an individual fibril basis. Using this method, we establish relative rate constants of collagen fibril assembly as functions of both temperature and concentration and determine the activation energy of self-assembly. Additionally, we find there is a linear relationship between collagen concentration and rate of fibril width growth within the concentration range used.