(485bj) Visual Observation of Linear Insulin Fibril Growth Conference: AIChE Annual MeetingYear: 2009Proceeding: 2009 AIChE Annual MeetingGroup: Food, Pharmaceutical & Bioengineering DivisionSession: Poster Session: Bioengineering Time: Wednesday, November 11, 2009 - 6:00pm-8:00pm Authors: Anand, G., Rensselaer Polytechnic Institute Belfort, G., Rensselaer Polytechnic Institute Amyloid diseases, including Alzheimer's disease and Parkinson's disease, are characterized by aggregation of normally functioning proteins into ordered fibrils. The proteins must lose their native structure and form beta-sheet structures that associate into long linear fibrils. The growth of the fibrils is often monitored by scattering techniques that give a bulk estimate of the average size of each particle. We have observed the growth and seeding of insulin fibrils under amyloid forming conditions using total internal reflection fluorescence (TIRF) microscopy. By using a dual-labeling construct, the original fibril seeds can be observed in a grown fibril. This was accomplished by tagging seed fibril covalently with a fluorescent dye (Alexa Fluor 568). This seed fibril was then added to a reaction of monomeric insulin. The elongated seed material was then contacted with thioflavin T, a fluorescent dye that detects the presences of amyloid fibrils. The entire fibril was observed at one excitation wavelength (488 nm), whereas the seeds are observed with a different excitation wavelength(569 nm). This allows the observation of fibril growth from a seed that is used to nucleate the elongation reaction. Some observations were that the seeds tend to be on the end of a fibril, indicating that the fibril only grows in unidirectionally, not in both longitudinal directions, as it is often depicted. Also, not all seeds have fibrils extended from them. And some fibrils have been nucleated without the presence of an observed preformed seed. This leads to a different model for fibril growth and nucleation than has been proposed earlier. This system is unique in that the fibril growth occurs in solution. Other studies that have used microscopy to observe fibril growth have first deposited the original seed material onto a solid substrate to observe fibril growth. This may affect the way that the fibril is formed. We have allowed the fibrils to form in solution and observed them after fibril formation is completed. In this fashion, the mechanism of fibril elongation in solution can be better studied.