Abstract
of the plane of focus, because patches in the process of disappearing invariably were not found in the planes Summary immediately above or below the plane of the patch (Figure S1 ). The only other patch component known to be Cortical actin patches are dynamic structures required stationary is Bee1/Las17 (hereafter referred to as Bee1), for endocytosis in yeast [1] . Recent studies have an activator of the Arp2/3 complex; however, Bee1 shown that components of cortical patches localize patches have a longer lifespan than Myo5 patches ([2] to the plasma membrane in a precisely orchestrated and Figures 1B and 1C) . Hence, the dynamics of Myo5 manner, and their movements at and away from the are different from all other patch proteins described to plasma membrane may define the endocytic memdate. brane invagination and vesicle scission events, reGiven the intriguing pattern of Myo5 localization, we spectively [2]. Here, through live-cell imaging, we anawent on to examine how it relates to the dynamics of lyze the dynamics of the highly conserved class I Bee1 and the Arp2/3 complex, because these two comunconventional myosin, Myo5, which also localizes to ponents represent different stages of actin patch matucortical patches and is known to be involved in endo- We set out to isolate temperature-sensitive mutants cued growth (data not shown). All experiments with of myo5 that could be used for examining the role of Myo5-GFP in this paper were performed in the doubleMyo5 in cortical patch dynamics. One particularly useful null background unless otherwise noted. We found that mutant carried a Glu-to-Ala substitution (E901A) in a Myo5-GFP exhibits rapid association and dissociation conserved sequence of the tail homology 1 (TH1) domain with cortical patches with an average lifespan of 13.1 Ϯ (Figure 2A ). This mutant (in the ⌬myo3 ⌬myo5 back-2.3 s (n ϭ 72) at 30ЊC ( Figures 1A, 3B, 3C , and Movie 1 ground) is temperature sensitive: the cells grew well at room temperature, slowly at 30ЊC, and did not grow at 37ЊC (Figure 2B ). The mutant protein was expressed

at a normal level and remained stable at the restrictive osin mutant strain, tpm1-2 ⌬tpm2, which is slow growing and has depolarized Myo5/actin patches at 30ЊC ([9], temperatures ( Figure S3 ).

We first characterized the localization and dynamics data not shown). As shown in Figure 3C , Myo5 patches in the tropomyosin mutant had a normal patch lifespan, of the mutant Myo5 E901A -GFP patches in the ⌬myo3 ⌬myo5 background. The Myo5
E901A
-GFP patches were suggesting that depolarization does not automatically result in an increased Myo5 patch lifespan. polarized at room temperature but depolarized at the semipermissive temperature 30ЊC (data not shown and There are two possible reasons for the increased lifespan of Myo5 E901A -GFP patches: (1) the mutation directly Figure 3A ). The same pattern was seen for actin patch localization in the mutant strain (data not shown). Timeprevents efficient release of Myo5 from the plasma membrane, or (2) the mutant Myo5 is maintained on the lapse analysis of the Myo5
E901A
-GFP patches showed that the mutant myosin I had an increased lifespan at cortex longer due to delayed completion of a function that requires Myo5. To distinguish between these possi-30ЊC with an average lifespan of 24.5 Ϯ 9.5 s (n ϭ 93) compared to 13. -GFP patches to normyo5 E901A mutant was a secondary defect resulting from depolarized Myo5/actin patches, we utilized a tropomymalcy ( Figure 3C ). This suggests that the prolonged patch lifespan in the myo5 E901A mutant strain is due to a delayed completion of a Myo5-dependent function.
E901A
To examine the effects of the myo5 E901A mutation on the dynamics of the Arp2/3 complex, two-color movies were made of a ⌬myo3 ⌬myo5 strain expressing Myo5 E901A -GFP and Arp2-DsRed at 30ЊC. In the mutant cells, many patches never exhibited colocalization of Myo5 and Arp2 and therefore might be abortive/aberrant structures (Movie 5). We reasoned that the primary defect in these cells could be found by analyzing patches to which both proteins localized and showed all phases of movement. While the myo5 E901A mutation had no effect on the rate of Arp2 fast movement, the slow movement phase of Arp2 was clearly prolonged in the mutant with an average duration of 15.9 Ϯ 5.6 s (n ϭ 100) compared to 9.4 Ϯ 2.3 s (n ϭ 100) in the wild-type. Student's t test showed that this difference was highly statistically significant (t ϭ 10.738 and p Ͻ 0.0001). Representative graphs of movement of individual Arp2 patches in the wild-type and the mutant are shown in Figure 3D . These plots also show that the Arp2 patches in the mutant often moved back and forth during their slow phase as though they were under force but unable to detach. These results suggest that a dysfunctional Myo5 leads to a delay in the initiation of the fast-movement phase of the Arp2/3 complex.
E901A
To further examine the functional significance of the abnormal patch dynamics in the myo5 E901A mutant, fluid phase endocytosis was assayed in these mutants with lucifer yellow (LY). Whereas wild-type cells efficiently internalized and accumulated LY in the vacuole, LY uptake was drastically reduced in the mutant cells at 30ЊC 

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