Effects of Actin Crosslinking Proteins on Actin Network RemodelingSumana Shashidhar, Alec Hoyland, and Sean Guo

Dept. of Quantitative Biology, Brandeis University, Waltham, MA 02454

Introduction Methods

DiscussionOur experiments conclusively determine that Abp1 and Srv2 act as crosslinking proteins, consolidating actin networks. We demonstrate that the actin filaments coalesce into crosslinked fibers, producing a denser structure. Our quantitative analysis demonstrated significant effects of each protein individually and in tandem. We found that the effect of Abp1 compounds with Srv2 producing a multiplicative model of reduced returns. At the final time point (t = 1200 s) the area is represented:

This is the formula for calculating the fold-change given two statistically independent operations, indicating that the mechanisms of Abp1 and Srv2 are biochemically independent, but that interaction with the same substrate alters the crosslinking efficiency of each protein species.

Results

The protein actin forms many structures with various functions that are central to cell functioning. Actin plays an important role in biological processes such as movement, the transportation of vesicles and organelles, endocytosis, cell division, and sensing the environment [1]. These processes are dependent upon the interactions between actin filaments and other proteins that aid in crosslinking and forming actin networks such as actin patches and the contractile ring [1].

Actin-binding protein 1 (Abp1) and suppressor of Ras-Val19 (Srv2) are two of these crosslinking proteins that enable actin to form networks or bundles and play a role in cellular activities. This project studies these proteins in a system constructed with actin, so that the networks that Abp1 and Srv2 create can be analyzed in vitro. Better understanding how these crosslinking proteins form actin networks allows for the mechanisms of many biological processes to be better defined. A basis for the development of hypotheses and models is then developed.

Abp1 is known to dimerize [2]. Srv2 is known to hexamerize [3]. We hypothesize that this polymerization leads to the cross-linking ability of Abp1 and Srv2, directly influencing actin dynamics. We utilized photobleaching and total internal reflection fluorescence (TIRF) microscopy to visualize actin dynamics in real time.

Sources[1] Pollard, Thomas D. and John A. Cooper. "Actin, a Central Player in Cell Shape and Movement." Science 326, no. 5957 (2009): 1208-1212. Accessed March 10, 2016. [2] Woo E-J, Marshall J, Bauly J, et al. Crystal structure of auxin-binding protein 1 in complex with auxin. The EMBO Journal. 2002;21(12):2877-2885.[3] Chaudhry F, Breitsprecher D, Little K, Sharov G, Sokolova O, Goode BL. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin. Pollard TD, ed. Molecular Biology of the Cell. 2013;24(1):31-41.

Protein Analysis: Concentration

Question

We expressed a recombinant protein in E. coli through the transfection of genes via antibiotic plasmids attached to the lac operon. The proteins were purified from the cell lysate by biochemical methods. Column chromatography; His-tag affinity was used to purify Srv2. To determine concentration, two methods were used. Spectrophotometric methods analyzed the absorbance of aromatic rings using known values and Beer’s Law. SDS-PAGE was also performed on the proteins to assay purity. Oligomeric state was assessed by single molecule photobleaching using TIRF microscopy. Real-time analysis of actin network remodeling in the presence of Abp1 and Srv2 was then assessed using TIRF microscopy. 10% of actin filaments were labeled with fluorophores in a microfluidic chamber. Each protein was flowed in individually. In addition, a trial with equal half concentrations of each protein was performed.

Special ThanksWe would like to thank Avital Rodal and Bruce Goode and their dogs.

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Fig. 1 Fig. 3

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PhotobleachingFig. 1 and 2 indicate the photobleaching steps as indicated by Heaviside drops in fluorescence over time. Fig 3 shows the initial fluorescence of Abp1 and Fig. 4 the fluorescence at t = 190 s.Due to incomplete binding of fluorophores to proteins and Boltzmann dissociation of polymers, we observed incomplete full polymerization. The maximum polymeric Markov state accounting for experimental error indicates the maximal polymerization state for the protein: dimer for Abp1 and hexamer for Srv2.Over time, fluorophores “burn out” and cease illumination. This process is temporally-correlated and random, allowing us to observe quantized drops in radiant flux of individual protein polymers.

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Quantitative AnalysisFig. 8 and 9 measure the area of the viewing plate upon which actin fluoresces above the noise threshold. Actin is shown to crosslink. Fig. 11 and 12 show the mean intensity of the fluorescence.The area of illumination decreases as the crosslinking proteins aggregate the actin into crosslinked polymeric fibers. The radiant flux increases as the area above threshold decreases due to crosslinking. These quantitative measures indicate the crosslinking capabilities of Abp1 and Srv2. Fig. 10 measures the normalized area of both proteins acting in concert and Fig. 13 measures the normalized mean intensity.The normalized area and mean of both proteins acting together obeys probabilistic laws of independence.

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Fig. 5, 6, and 7. Apb1, Srv2, and a 1:1 mixture acting on the actin network in silicoActin DynamicsFig. 5 and 6 visualize a montage of the actin network, with an image taken every 20 seconds under TIRF microscopy. Both proteins crosslink actin filaments, producing thickened fibers. Fig. 7 shows the action of Abp1 and Srv2 acting together in a 1:1 mixture.

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Method 1: SDS – PAGE Method 2: Spectrophotometry

After denaturing the protein and placing diluted protein samples in the gel, the molecular weight can be determined through comparison with the ladder as seen in Fig. 14. Impurities are separated and purity of the protein can be assessed.

Fig. 14

Determining the absorbance of 2uL samples of the Abp1 and Srv2 at 280 nm allowed for concentration to be calculated using the known extinction coefficient and pathway length. Impurities are not separated and counted within the concentration.

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Srv2: Normalized Mean Radiant Flux above Threshold

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