Robust polarity establishment occurs via an endocytosis-based cortical corralling mechanism

Abstract

Formation of a stable polarity axis underlies numerous biological processes. Here, using high-resolution imaging and complementary mathematical modeling we find that cell polarity can be established via the spatial coordination of opposing membrane trafficking activities: endocytosis and exocytosis. During polarity establishment in budding yeast, these antagonistic processes become apposed. Endocytic vesicles corral a central exocytic zone, tightening it to a vertex that establishes the polarity axis for the ensuing cell cycle. Concomitantly, the endocytic system reaches an equilibrium where internalization events occur at a constant frequency. Endocytic mutants that failed to initiate periodic internalization events within the corral displayed wide, unstable polarity axes. These results, predicted by in silico modeling and verified by high resolution in vivo studies, identify a requirement for endocytic corralling during robust polarity establishment.

Figure 1.

Schematics illustrating the mathematical model. Shown are the Cdc42 autoamplification module (A), the complete endocytosis module (B), the exocytosis module (C), and a legend for graphics (D).

Figure 2.

Robust polarity establishment involves dynamic changes in endo- and exocytic trafficking systems. (A) Transition of a typical in silico WT cell from nonpolarized to a polarized state. Membrane-bound Cdc42-GTP depolarized over the plasma membrane (top left) polarizes to a unique cluster over time (top right). The Cdc42 kymograph (top) shows Cdc42-GTP during polarization. The kymograph in the bottom shows individual endo- and exocytic events over time (x axis) along the cortex (y axis). A tight pole of exocytosis develops (cyan), overlapping the Cdc42-GTP cluster, and is corralled by a ring of endocytosis (red). (B) Random endo- and exocytic distributions in vivo in a nonpolarized cell (left) change to an organized “bull’s-eye pattern” in a polarized cell (right) with a tight exocytic zone surrounded by endocytic vesicles. The endo- and exocytic zones are marked by Abp1-RFP (red) and GFP-Sec4 (cyan), respectively. Kymographs represent the endo- and exocytic profiles along the cortex. Bud emergence occurs at the end of the kymographs. Bars, 2 µm. (C) In silico and in vivo statistical analyses of the coincident tightening of the exocytic (cyan) and endocytic (red) zones as WT cells polarize with the SD between cells represented by error bars.

Figure 3.

Robust polarity establishment involves the generation of a specific endocytic signature. (A) Kymograph of a polarizing WT cell expressing GFP-Sec4 and Abp1-RFP markers (left). Endocytic dynamics evolve from a series of discrete random events (left) to a pattern, or signature, of constant frequency and amplitude as the cell becomes stably polarized (right). The temporal change in intensity of the whole cell (black) was negligible compared with endocytic pattern at the cell cortex (red). (B) The temporal change in the intensity of random endocytic events of a nonpolarized cell (gray) versus regular events of constant amplitude in a polarized cell (red). (C) A scatter dot plot of the in silico (left) and in vivo (right) data comparing the frequency of endocytic events above the threshold (M&M) in nonpolarized (gray) versus polarized (red) cells. The black bars indicate the mean and SD over N events.

Figure 4.

Endocytic cortical corralling is required for robust polarity establishment. The endo- and exocytic vesicles are marked by Abp1-RFP (red) and GFP-Sec4 (cyan), respectively. (A, top) The kymograph of an in silico sla2Δ mutant cell shows depolarized endocytic events throughout the simulation, whereas exocytic clusters were unstable in the absence of corralling. (bottom) In vivo analysis shows polarizing sla2Δ cells to exhibit single or multiple exocytic poles with randomly distributed endocytic sites. Bars, 2 µm. (B) Abp1-RFP (closed circle) and GFP-Rvs167 (open circle) residency times in actin patches for WT (red) and sla2Δ (purple) cells, represented by a scatter dot plot. The black lines indicate the mean and SD over N patches. Mean residency times of the proteins were significantly different between WT and sla2Δ cells (P < 0.0001). (C) The temporal change in the intensity of endocytic events reveals a random endocytic pattern for polarized sla2Δ cells (purple) in contrast to polarized WT cells (red). (D) A scatter dot plot depicting the frequency of endocytic events above the threshold in polarized endocytic mutants such as sla2Δ (purple), sla1Δ bbc1Δ (blue), and rvs167Δ rvs161Δ (pink) versus polarized WT cells (red). The black bars indicate the mean and SD over N events.

Figure 5.

Endocytic cortical corralling is required for focused exocytic pole formation. Endo- and exocytic vesicles are marked by Abp1-RFP (red) and GFP-Sec4 (cyan), respectively. (A) In silico and in vivo analyses of the distributions of exocytic cluster size for polarized WT cells (red) compared with endocytic mutants such as sla2Δ (purple), sla1Δ bbc1Δ (blue), rvs167Δ rvs161Δ cells (pink), ede1Δ (black), and clc1Δ (orange) cells. The mean pole diameter is significantly larger in mutants (except for in silico rvs167Δ rvs161Δ) compared with WT (P < 0.01). The black bars indicate the mean and SD. (B) Kymograph of polarized WT cells in vivo (top) displays focused exocytic pole (cyan) during polarization corralled by endocytic vesicles (red). Endocytic mutants defective in endocytic corralling such as sla1Δ bbc1Δ (bottom) and rvs167Δ rvs161Δ (middle) display depolarized endocytic vesicles (red) and wider exocytic poles (cyan).