A catalytic role for Mod5 in the formation of the Tea1 cell polarity landmark

Abstract

Many systems regulating cell polarity involve stable landmarks defined by internal cues. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5. Tea1 and Mod5 are highly concentrated in clusters at cell tips in a mutually dependent manner, but how the Tea1-Mod5 interaction contributes mechanistically to generating a stable landmark is not understood. Here, we use live-cell imaging, FRAP, and computational modeling to dissect dynamics of the Tea1-Mod5 interaction. Surprisingly, we find that Tea1 and Mod5 exhibit distinctly different turnover rates at cell tips. Our data and modeling suggest that rather than acting simply as a Tea1 receptor or as a molecular "glue" to retain Tea1, Mod5 functions catalytically to stimulate incorporation of Tea1 into a stable tip-associated cluster network. The model also suggests an emergent self-focusing property of the Tea1-Mod5 cluster network, which can increase the fidelity of polarized growth.

Figure 1

Mod5 Turns over More Quickly Than Tea1 within Cell Tips (A) Localization of Tea1-GFP and GFP-Mod5 in wild-type and tea1Δ cells, showing regions bleached in full-tip and half-tip FRAP experiments. Scale bar represents 5 μm. Fluorescence recovery of (B) Tea1-GFP after full-tip (n = 20) and half-tip (n = 13) bleaching in wild-type cells; (C) GFP-Mod5 after full-tip bleaching in wild-type (n = 16) and tea1Δ (n = 19) cells; (D) GFP-Mod5 after full-tip and half-tip (n = 19) bleaching in wild-type cells. GFP-Mod5 shows increased recovery after half-tip bleaching. For clarity the same full-tip trace is duplicated in (C) and (D). Error bars show standard deviations. See also Figure S1 and Table S1.

Figure 2

Interaction of Tea1 and Mod5 in the Formation of Tea1 Cluster Networks (A) Images and kymographs showing delivery of Tea1-GFP, Tea1Δdimer-GFP, and Tea1Δtrimer-GFP to cell tips. Tea1Δtrimer fails to accumulate at cell tips. (B) Steps in Tea1 polymerization and Mod5 dynamics. Incoming Tea1 on microtubules is shown in light blue, with a microtubule in green. Tea1 associated with cluster networks is shown in dark blue. Mod5 is shown in red. Mod5 diffuses in the membrane (i) and promotes the incorporation of newly arrived Tea1 (ii, iii) but remains restricted to the tip region by diffusion-capture mechanism (iv). (C) Proposed interactions between Tea1 and Mod5 (i), and between Tea1 and Tea1 (ii), with associated rate constants. Tea1 associated with membranes but not with cluster networks is shown in light blue. (D) Kinetic scheme used for computational modeling. (E) Conceptual diagram of temporal evolution of Tea1 cluster network as a result of the creation and dissolution of the Tea1-Tea1 and Tea1-Mod5 bonds described in (C). Further details are in text. See also Figure S2 and Movie S1.

Figure 3

In Silico Simulations Recapitulate Tea1 Landmark Formation and Accurately Predict System Dynamics (A–D) Time evolution of Tea1 cluster-network formation at cell tips, showing local concentration of Tea1 incorporated into cluster networks (Tpol), Mod5 bound to cluster networks (Mpol), and free Mod5 (Mmem). The x axis indicates distance along cell perimeter. (E) Population-averaged distribution of Tea1-GFP at cell tips in wild-type cells, calculated from 60 individual images. Green dots indicate positions of Tea1-GFP deposition by microtubules from video sequences (211 events, from eight cell tips). Note Tea1 deposition events far from the cell tip center. (F) In silico steady-state Tea1 distribution. (G) Normalized steady state Tea1-GFP fluorescence at the cell tip, measured from (E) (dashed line) and (F) (solid line). Shaded area represents 95% confidence interval for model prediction (see Experimental Procedures). (H) Predicted in silico and experimental Tea1:Mod5 cell tip ratios. Values without parentheses refer to total Tea1 and Mod5 at tips, and values in parentheses refer to cluster-network-associated Tea1 and Mod5 at tips. (I–L) Fluorescence recovery after full-tip photobleaching of (I) Tea1-GFP (n = 8), (J) GFP-Mod5 (n = 13), (K) Tea1-GFP (n = 11), and (L) GFP-Mod5 (n = 11) in cells with altered Mod5 expression. In (I)–(L), solid lines represent in silico predictions and dots represent in vivo measurements. Shaded areas indicate confidence intervals of model predictions estimated from parameter variation (Experimental Procedures). For comparison, data (dots) and model simulations (lines) for wild-type Mod5 expression (Figure S3) are shown in gray. See also Figure S3 and Table S2.

Figure 4

Tea1 Cluster Networks Exhibit Self-Focusing Behavior (A and B) Directionality of Tea1 cluster-network polymerization depends on the local concentration of polymeric Tea1. In regions with high polymeric Tea1, autocatalysis drives net Tea1 polymerization, and the Mod5 “cycle” proceeds in a clockwise direction (A; green zone in D). In regions with low polymeric Tea1 there is net Tea1 depolymerization, and the Mod5 cycle proceeds anticlockwise (B; red zone in D). (C) Normalized concentration of polymeric Tea1 during de novo network formation as predicted by the model, shown together with the normalized profile of Tea1 deposition by microtubules. The cluster network becomes progressively more focused over time. The x axis indicates distance along cell perimeter. (D) Normalized Tpol concentration and polymerization reaction flux at steady state. The x axis is as in (C). See also Figure S4.