Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces

Abstract

Many organisms are protected from freezing by the presence of extracellular antifreeze proteins (AFPs), which bind to ice, modify its morphology, and prevent its further growth. These proteins have a wide range of applications including cryopreservation, frost protection, and as models in biomineralization research. However, understanding their mechanism of action remains an outstanding challenge. While the prevailing adsorption-inhibition hypothesis argues that AFPs must bind irreversibly to ice to arrest its growth, other theories suggest that there is exchange between the bound surface proteins and the free proteins in solution. By conjugating green fluorescence protein (GFP) to a fish AFP (Type III), we observed the binding of the AFP to ice. This was accomplished by monitoring the presence of GFP-AFP on the surface of ice crystals several microns in diameter using fluorescence microscopy. The lack of recovery of fluorescence after photobleaching of the GFP component of the surface-bound GFP-AFP shows that there is no equilibrium surface-solution exchange of GFP-AFP and thus supports the adsorption-inhibition mechanism for this type of AFP. Moreover, our study establishes the utility of fluorescently labeled AFPs as a research tool for investigating the mechanisms underlying the activity of this diverse group of proteins.

FIGURE 1

Ribbon diagram of the Type III antifreeze protein (AFP) linked through its N-terminus to the C-terminus of green fluorescence protein (GFP). The light blue region is the ice-binding site. The α-helices are shown in red and β-strands in green.

FIGURE 2

Experimental cell. A schematic drawing of the temperature-controlled cell. Details in text.

FIGURE 3

Ice crystals in the presence of GFP-AFP type III. Ice crystals were produced in a solution containing GFP-AFP and a free dye (Cy5-dUTP) and imaged with 488 nm and 633 nm illumination lines through two separate fluorescence filters (a Cy5 filter and a GFP filter). Row A displays images of ice crystals representing (A1) both GFP and Cy5 fluorescence; (A2) GFP fluorescence; (A3) Cy5 fluorescence; and (A4) subtraction of the Cy5 image from the GFP image according to Eq. 1. Row B: Magnified image of the boxed crystal in A1. (B1) The bright fluorescence in the middle of the crystal (core) corresponds to ice formed during the fast growth phase, whereas ice grown slowly during reshaping to the bipyramidal structure (peripheral ice) has lower fluorescence intensity. Row C: Model of the bipyramidal ice crystal shape and a three-dimensional Gaussian PSF (C1) and the convolutions between them: (C2) weighted sum of surface and solution, (C3) solution only, and (C4) crystal only. These simulations did not include the contribution from the core. Row D shows the molecules that are present: GFP, green circles; and Cy5, red circles. Solid circles represent molecules detected by fluorescence with a particular optical filter and open circles denote molecules that are not detected. AFP domains are blue.

FIGURE 4

Unconjugated GFP does not accumulate within or on the surface of ice crystals. An ice crystal was grown in a solution containing AFP, unconjugated GFP, and Cyanine 5-dUTP. The ice crystal was illuminated with 488 nm and 633 nm lasers and imaged through a GFP filter (A) and through a Cy5 filter (B). For both filters the crystal appears dark. Panel C shows the outcome of the subtraction of the I_Cy5 image from the I_GFP image. The intensity of fluorescence along the blue line in the subtracted image is displayed in the graph (D). The lower parts of panels (A–C) show the molecules that are present as described in the caption for Fig. 3.

FIGURE 5

FRAP experiment. Confocal images of crystals recorded over 20 h. For each of the displayed crystals, half of the crystal was bleached and half was left unbleached. The intensities of the bleached and unbleached parts were monitored as a function of time for a period of 20 h. The average signals from the bleached parts of ∼20 crystals, as well as those from the unbleached parts, are shown in Fig. 6.

FIGURE 6

Absence of exchange of ice-bound fluorescent antifreeze proteins after photobleaching. This graph shows the results from the FRAP experiments. The surface intensities of the bleached (circles) and unbleached (triangles) regions of crystals were calculated according to Eq. 3 and displayed as a function of time. Kinetic models of the recovery of the fluorescence signal after photobleaching with recovery periods of one day (purple line) and seven days (red line for bleached and orange line for unbleached) are also shown. Bleaching of 2% per observation is included in the model. The data were averaged over several crystals in four separate experiments. The number of crystals n at each time window was (0 ≤ t ≤ 14 h, 20 ≤ n ≤ 27), (5 ≤ t ≤ 14 h, 17 ≤ n ≤ 19), and (15 ≤ t ≤ 20 h, 9 ≤ n ≤ 14) for the bleached regions; and (0 ≤ t ≤ 10 h, n = 12), (11 ≤ t ≤ 14 h, 9 ≤ n ≤ 10), and (15 ≤ t ≤ 16 h, n = 2) for the unbleached regions.

FIGURE 7

Photobleaching and recovery after reshaping of ice. Row A contains the summation of GFP and Cy5 fluorescence images of an ice crystal as in Fig. 3 A1. Row B shows the corresponding subtracted images as described in Eq. 1 and Fig. 3 A4. Column 1 corresponds to the initial image before bleaching. Column 2 corresponds to the crystal after photobleaching. The fluorescence intensity did not recover within the experimental period of several hours (see Fig. 6). Column 3 corresponds to the same crystal after it had been warmed to slightly above its melting temperature and then cooled to allow reshaping to the bipyramidal shape. GFP-AFP was found to accumulate on the newly formed surfaces of the crystal.