Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features

Abstract

Conserved proteins drive clathrin-mediated endocytosis (CME), which from yeast to humans involves a burst of actin assembly. To gain mechanistic insights into this process, we performed a side-by-side quantitative comparison of CME in two distantly related yeast species. Though endocytic protein abundance in S. pombe and S. cerevisiae is more similar than previously thought, membrane invagination speed and depth are two-fold greater in fission yeast. In both yeasts, accumulation of ~70 WASp molecules activates the Arp2/3 complex to drive membrane invagination. In contrast to budding yeast, WASp-mediated actin nucleation plays an essential role in fission yeast endocytosis. Genetics and live-cell imaging revealed core CME spatiodynamic similarities between the two yeasts, although the assembly of two zones of actin filaments is specific for fission yeast and not essential for CME. These studies identified conserved CME mechanisms and species-specific adaptations with broad implications that are expected to extend from yeast to humans.

Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

“…In the wsp1ΔCA mutant the average time between the first appearance of Fim1-GFP and when it reaches peak concentration, which we refer to here as the Fim1 assembly time, increased from 5.1 to 6.7 seconds, consistent with another recent study (Fig. 1B,C) (Sun et al, 2019). In addition, the wsp1ΔCA mutation decreased the percentage of actin patches that internalized (Fig 1F, VideoS3).…”

“…Each of these NPFs is relatively potent in activating Arp2/3 complex in vitro , and analysis of wsp1 mutant or dip1 knockout strains suggests that activation of Arp2/3 complex by both NPFs is required for normal endocytic actin assembly (Basu and Chang, 2011; Sirotkin et al, 2005; Wagner et al, 2013). On the other hand, while multiple experiments suggest the motor activity of Myo1 is important for normal actin dynamics, mutations in the Myo1 Arp2/3-activating segment do not influence endocytic internalization or coat protein dynamics, indicating the NPF activity of Myo1 may not by important for actin assembly in S. pombe (MacQuarrie et al, 2019; Sun et al, 2019).…”

“…At sites of endocytosis in S. pombe , Arp2/3 complex nucleates the assembly of branched actin networks that drive invagination of the plasma membrane (Sun et al, 2019). The activity of Arp2/3 complex at endocytic sites can be controlled by at least three distinct NPFs: Wsp1, Dip1, and Myo1 (Sirotkin et al, 2005; Wagner et al, 2013).…”

“…For instance, in dip1Δ strains, the rate of initiation of new patches is markedly decreased, but once an endocytic actin network is initiated, it assembles rapidly, suggesting Dip1 is important for seeding but not propagation of the network (Basu and Chang, 2011). Further, deletion of the Wsp1 CA segment motif causes failure of endocytic actin patches to internalize, a process thought to be dependent on the propagation of branches (Sun et al, 2019). However, some data suggests that the seeding and propagating roles of Wsp1 and Dip1 might not be distinct.…”

Spatial regulation of clathrin-mediated endocytosis through position-dependent site maturation

“…While we did not calibrate fluorescence intensity to count molecules, relative fluorescence intensities we measured are in agreement with those reported previously (Fig. S1A, Tables S5-7; Picco et al, 2015; Sun et al, 2019). The median lifetimes we measured are also in general agreement with published results (Fig.…”