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. 2006 May;17(5):2439-50.
doi: 10.1091/mbc.e05-11-1015. Epub 2006 Feb 22.

FUS1 regulates the opening and expansion of fusion pores between mating yeast

Scott Nolan  1 , Ann E Cowan, Dennis E Koppel, Hui Jin, Eric Grote
Affiliations

FUS1 regulates the opening and expansion of fusion pores between mating yeast

Scott Nolan et al. Mol Biol Cell. 2006 May.

Abstract

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.

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Figures

Figure 1.
Figure 1.
Cell fusion detected by the exchange of fluorescent markers. (A) Cytoplasmic mixing. MATa cells expressing cytoplasmic GFP were mated with MATα cells expressing cytoplasmic DsRed and monitored by time-lapse microscopy (Supplementary Movie 1). (B) Slow transfer of a plasma membrane protein. MATa SSO2-GFP cells were mated to MATα DsRed cells and monitored by time-lapse microscopy. Sso2-GFP diffused slowly into the MATα cell and was absent from emerging buds (arrowheads).
Figure 2.
Figure 2.
Permeance calculation for a typical fusion pore. The permeance equation, P(t) = [VDVR/(VD + VR)]dln[ID(t) - IR(t)(VD/VR)]/dt, was derived from Fick's law of diffusion. (A) Fluorescence intensity measurements. A series of fluorescent images of a field of mating MATa GFP and MATα cells was collected at 2.5-s intervals. Boundaries were drawn around the two cells of a mating pair and the mean fluorescence intensity in each cell was measured for each image. To control for photobleaching and other extrinsic factors, fluorescence intensity was also measured for a set of adjacent MATa GFP cells that did not fuse and for a background region. (B) The raw fluorescence intensity data were corrected for background fluorescence and photobleaching and then multiplied by the area of the cell. (C) The volume adjusted intensity difference between the cells approaches 0 as GFP diffuses to equilibrium. (D) The natural log of the volume adjusted intensity difference was calculated to fit the form of the permeance equation shown above. (E) A second-order polynomial equation was fit to the logarithmic data for the interval corresponding to the start of GFP movement until GFP approached equilibrium. (F) The fitted curve was multiplied by a volume constant to yield permeance in μm3/s.
Figure 3.
Figure 3.
Abrupt expansion (A), and transient opening (B) of fusion pores between wild-type mating yeast. Top, GFP and DsRed fluorescence intensity measurements for the two cells of a mating pair. Bottom, the logarithms of the volume adjusted intensity differences were calculated as shown in Figure 2D and then fit with second-order polynomial curves. The accuracy (R2 value) of each fit is displayed. In A, permeance abruptly expands after 169 s. In B, a small pore opens at 16 s and closes at 234 s. A larger permeance flux initiates at 477 s. Note that DsRed transfers later and more slowly than GFP because it is a 108-kDa tetramer.
Figure 4.
Figure 4.
Theoretical relationship between GFP permeance and the fusion pore radius.
Figure 5.
Figure 5.
Osmotic regulation of pore permeance. MATa GFP cells and MATα cells were grown and then mated in media supplemented with 1 M sorbitol (n = 20) or under standard conditions (n = 19). Mean initial permeances and rates of permeance increase were calculated. Error bars, SEM. p values are from a Student's t test.
Figure 6.
Figure 6.
Fusion pore permeance in cell fusion mutants. (A) prm1. Fusion pore permeance was measured after a hypoosmotic shift for wild-type (n = 19) and prm1 (n = 18) mating pairs. (B) Cell wall remodeling mutants. Fusion pore permeance was measured under standard conditions for wild-type (n = 32), fus1 (n = 31), fus2 (n = 37), rvs161 (n = 41), and spa2 (n = 47) mating pairs.
Figure 7.
Figure 7.
Persistently slow expansion of fus1 fusion pores. (A) Monitoring late stages of fusion pore expansion with Gag-GFP. GFP was fused to the structural protein of the L-a virus. MATa cells expressing Gag-GFP were mated to MATα mCherry cells. Cytoplasmic mixing was followed by time-lapse microscopy. (B) Delayed initiation of Gag-GFP transfer in fus1 mating pairs. The time interval between the initiation of plasma membrane fusion marked by mCherry transfer and the time point when the pore had expanded sufficiently to detect Gag-GFP transfer was compared for wild-type (n = 22) and fus1 (n = 21) fusion pores. (C) Slow Gag-GFP transfer in fus1 mating pairs. After the initiation of Gag-GFP transfer, the time required for 33% of the mobile fraction of Gag-GFP to diffuse across the fusion pore was compared for wild-type (n = 22) and fus1 (n = 19) fusion pores. Gag-GFP did not reach equilibrium before the end of the recorded interval in 2 fus1 mating pairs.
Figure 8.
Figure 8.
Late mating events are delayed in fus1 mating pairs. (A) Detecting karyogamy and vesicle transfer. MATa cells expressing Hmg1-GFP were mated to MATα DsRed cells. At various time points, mating was arrested by transferring the yeast to azide buffer at 4°C. Mating pairs were then stained with DAPI and observed by fluorescence microscopy. Cytoplasmic DsRed transferred after plasma membrane fusion. Hmg1-GFP transferred slowly between cells in ER derived transport vesicles and then rapidly diffused throughout the combined nuclear envelope after karyogamy. (B) Images from a time-lapse series documenting plasma membrane and vacuole fusion (Supplementary Movie 2). MATa GFP cells were pulse-labeled with FM4-64 to mark vacuole membranes and then mated to MATα cells containing CellTracker Blue CMAC-labeled vacuoles. Vacuoles were retained within their original parent until a diploid bud emerged 50 min after plasma membrane fusion. (C-F) Mating pairs that had completed plasma membrane fusion as evidenced by GFP or DsRed transfer were scored for Hmg1-GFP vesicle transfer (C), karyogamy (D), zytogic bud emergence (E), and vacuole fusion (F). The apparent reduction in zygotic budding at the 4-h time point in wild-type mating pairs results from separation of the first zygotic bud from the fused mating pair.
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