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. Author manuscript; available in PMC: 2013 Oct 16.
Published in final edited form as: Dev Cell. 2012 Oct 16;23(4):769–781. doi: 10.1016/j.devcel.2012.09.001

Myosin-V is activated by binding secretory cargo and released in coordination with Rab/exocyst function

Kirk W Donovan *, Anthony Bretscher *,1
PMCID: PMC3725570  NIHMSID: NIHMS407098  PMID: 23079598

Abstract

Cell organization requires motor-dependent transport of specific cargos along cytoskeletal elements. How the delivery cycle is coordinated with other events is poorly understood. Here we define the in vivo delivery cycle of myosin-V in its essential function of secretory vesicle transport along actin cables in yeast. We show myosin-V is activated by binding a secretory vesicle, and myosin-V mutations that compromise vesicle binding render the motor constitutively active. About 10 motors associate with each secretory vesicle for rapid transport to sites of cell growth. Once transported, the motors remain associated with the secretory vesicles until they undergo exocytosis. Motor release is temporally regulated by vesicle-bound Rab-GTP hydrolysis and requires vesicle tethering by the exocyst complex, but does not require vesicle fusion with the plasma membrane. All components of this transport cycle are conserved in vertebrates, so these results should be generally applicable to other myosin-V delivery cycles.

INTRODUCTION

Cell polarity enables cells to perform their physiological function and allows for growth and division. Overall cell polarity in eukaryotic cells is established by a polarized cytoskeleton, which is then used by motor proteins to transport various cargos to their proper destination (Goode et al., 2000). Much is known about how microtubules and microfilaments, the tracks for motor proteins, are assembled and how motor proteins such as kinesins, dyneins and myosins mediate cargo transport (Akhmanova and Hammer, 2010); however, little is known about how their transport cycles are coordinated with cargo association and delivery.

The class-V myosins are among the most evolutionarily conserved motor proteins and are responsible for transporting specific cargos in fungal, plant (called myosin-XI) and animal cells (Hammer and Sellers, 2011). Whereas kinesins transport cargos over longer distances, myosin-V motors function to transport cargo more locally along actin filaments that are often associated with the plasma membrane. For example, a specific splice isoform of vertebrate myosin-Va (MyoVa) associates with Rab27a and melanophilin to capture melanosomes involved in hair pigmentation (Wu et al., 2002); MyoVa also transports the endoplasmic reticulum into dendritic spines (Wagner et al., 2010). Additionally, myosin-Vb in association with Rab proteins, including Rab11 and Rab8, has been implicated in endocytic trafficking pathways (Lapierre et al., 2001; Hales et al., 2002; Roland et al., 2007). The importance of myosin-Vs in humans is underscored by the findings that defects in MyoVa cause Griscelli syndrome, and defects in MyoVb result in microvillus inclusion disease (Pastural et al., 1997; Müller et al., 2008). To perform these functions, all myosin-Vs consist of two heavy chains with N-terminal motor domains, a long lever arm containing six IQ motifs with associated light chains, a dimerization domain, and a C-terminal cargo-binding tail domain (Hammer and Sellers, 2011).

Budding yeast utilizes a myosin-V to transport its essential cargo of secretory vesicles very rapidly along polarized actin cables from the mother cell to sites of cell growth in the bud (Pruyne et al., 1998). This myosin-V, whose heavy chain is encoded by the essential MYO2 gene, is also involved in organelle segregation during the cell cycle (Weisman, 2006). Each cargo has a specific receptor that is recognized by the Myo2p tail, with vacuolar segregation depending on Vac17p (Ishikawa et al., 2003), peroxisome segregation on Inp2p (Fagarasanu et al., 2006), and nuclear orientation on Kar9p (Yin et al., 2000). As in vertebrate cells, Rab proteins bind to the tail of Myo2p to facilitate movement of secretory compartments. Ypt31/32p (the Rab11 homolog) transports late-Golgi compartments through GTP-dependent binding to Myo2p (Lipatova et al., 2008); similarly, the Rab8 homolog Sec4p participates in GTP-dependent binding of Myo2p to transport secretory vesicles to sites of growth. Transport of both of these compartments also requires the regulatory phospholipid PI4P (Santiago-Tirado et al., 2011).

Collectively, these studies show that myosin-Vs transport cargo in a receptor-mediated manner. Biophysical and biochemical studies have suggested that myosin-Vs can exist in a folded, inactive form in which the tail domain interacts with the head domain (Krementsov et al., 2004; Li et al., 2004; Wang et al., 2004; Liu et al., 2006; Thirumurugan et al., 2006), although currently little is known about how myosin-Vs might be activated in vivo. Therefore, major gaps in the current understanding include how association with cargo affects myosin-V function and how motor release is integrated with events at the delivery site. Yeast provides an especially amenable system to study these questions as vesicle capture in the mother is spatially segregated from sites of delivery and exocytosis in the bud; further, secretory vesicles are by far the most abundant cargo it delivers over its cell cycle (Pruyne et al., 2004).

After Myo2p transports secretory vesicles into the bud, the exocyst complex tethers the vesicle to the cortex before fusion can occur (He and Guo, 2009). During transport and in preparation for exocytosis, seven subunits of the exocyst (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p) associate with secretory vesicles by the interaction between GTP-bound Sec4p on the vesicle membrane and Sec15p in the complex (Guo et al., 1999; Boyd et al., 2004). These subunits then meet with cortex-localized pools of Exo70p and Sec3p to tether the vesicle to the plasma membrane (Boyd et al., 2004). Once tethered, downstream SNARE action between the v-SNARE Snc1/2p (synaptobrevin/VAMP) and t-SNAREs Sso1p (syntaxin) and Sec9p (SNAP-25) is believed to drive the fusion event (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994).

Here we show that Myo2p is activated through binding to secretory cargo, and then define the number of motors that transport vesicles to sites of growth. Further, we show that Myo2p remains associated with secretory cargo after arrival at sites of growth and its release from vesicles is coordinated with exocyst complex tethering and Sec4-GTP hydrolysis.

RESULTS

Myo2p is activated by binding transport-competent cargo

Myo2p has to deliver many organelles at appropriate times during the cell cycle. For the vacuole, timing is dictated by the cell cycle regulated synthesis and destruction of the vacuole receptor, Vac17p (Tang et al., 2003). Since Myo2p is polarized throughout the cell cycle in actively growing yeast, one model suggests that the motor is always active and transports any cargo for which a receptor is available. Alternatively, Myo2p might need to be activated by the presence of transport-competent cargo. To distinguish between these models, we examined Myo2p localization when transport-competent secretory cargo, Myo2p’s most abundant and essential cargo, is eliminated.

Myo2p was visualized in live cells by appending either 1xGFP or 3xGFP to the 3’-end of the MYO2 open reading frame in the chromosome, neither of which had any detectable effect on cell growth or Myo2p localization. We first examined Myo2–3GFP in wild-type cells and in cells harboring conditional mutations in essential secretory pathway genes. Sec23p is a component of COPII vesicles that transport secretory materials from the endoplasmic reticulum to the Golgi apparatus; in the conditional sec23-1 mutant all export from the ER is blocked at the restrictive temperature, thereby also preventing post-Golgi secretory vesicles from being formed (Figure S1a; Kaiser and Schekman, 1990; Rexach and Schekman, 1991). Sec4p is the Rab GTPase associated with secretory vesicles that functions as part of the receptor for Myo2p; in the conditional sec4-8 mutant secretory vesicles are made, but not transported at the restrictive temperature (Salminen and Novick, 1987; Ferro-Novick and Novick, 1993). In wild-type cells and the sec23–1 and sec4-8 mutants growing at the permissive temperature, Myo2–3GFP is polarized to sites of growth. However, upon shifting to the restrictive temperature for 30 minutes Myo2–3GFP became depolarized in both mutants whereas it remained polarized in wild-type cells (Figures 1A and 1C). This depolarization is not a result of an indirect defect of the actin cytoskeleton as normal actin cables were observed extending into the mother cell at both temperatures in all strains (Figure 1B). Thus, Myo2p must normally be activated by binding cargo.

Figure 1. Myo2p polarization requires competent secretory cargo.

Figure 1

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A. Polarization of Myo2–3GFP in wild-type, sec4-8, and sec23-1 at the permissive temperature and after a 30 min shift to the restrictive temperature. Images adjusted for best presentation. Scale bar, 5 µm. See also Figure S1a.

B. Phalloidin staining to show the actin cytoskeleton in the indicated strains. Scale bar, 5 µm.

C. Quantification of Myo2–3GFP polarization in (A). Three independent replicates of 150 small and medium buds were scored for each condition to determine the fraction of Myo2–3GFP polarized. Error bars are standard deviation.

D. Myo2-GFP and Myo2-13-GFP polarization in SEC23 and sec23-1 cells at 21°C and 35°C following a 30 minute shift. Images adjusted for best presentation. Scale bar, 5 µm. See also Figure S1b–h.

We have characterized conditional mutations in the Myo2p tail that fail to interact with and transport secretory vesicles at the restrictive temperature, but Myo2p nevertheless remains polarized (Schott et al., 1999, and Figure S1b–d). Since we now know that competent secretory cargo is necessary to polarize wild-type Myo2p, tail mutants at the restrictive temperature must be both defective in cargo binding and constitutively active. To test this model, we first introduced myo2–13-GFP into a sec23-1 strain. Myo2–13-GFP was polarized in both SEC23 and sec23-1 cells at 21° and 35°, thereby indicating that Myo2–13-GFP is active in the presence and absence of secretory cargo (Figure 1D). Further, this polarization is actin dependent and the recovery of Myo2–13-GFP into the bud after photobleaching follows wild-type recovery kinetics (Figure S1e–h); this indicates that Myo2–13-GFP polarization is due to motor activity. In summary, wild-type Myo2p needs to be activated to polarize, but a mutant defective in binding secretory vesicles is constitutively active and can polarize in the absence of cargo.

About 10 myosin-V motors associate with secretory vesicles

To investigate the delivery cycle of Myo2p, we first needed to quantify the number of motors in a yeast cell. Using quantitative immunoblots comparing purified GST-GFP with Myo2-GFP in total cell lysates, we estimate there are about 5800 Myo2-GFP molecules (2900 motors) per cell, which is in good agreement with a previous estimate (Figures 2A and B; Ghaemmaghami et al., 2003).

Figure 2. 10 Myo2p motors associate with vesicles undergoing active transport to sites of growth.

Figure 2

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A. Quantitative immunoblot of Myo2-GFP and GFP-Sec4 lysates. Standard curve of GST-GFP (0.5 – 20 ng) was loaded onto 6–10% SDS-PAGE gel with indicated volumes of Myo2-GFP and GFP-Sec4 lysate and blotted with GFP antibodies.

B. Number of molecules of Myo2-GFP and GFP-Sec4 per cell, as determined from quantitative immunoblots. Note that monomers of Myo2-GFP are shown. Error bars are standard deviations from three independent cell lysates for both Myo2-GFP and GFP-Sec4.

C. Myo2-4IQ-3GFP on RFP-Snc1 positive diffusing vesicles. Arrowheads show vesicle/motor puncta colocalization. Scale bar, 2 µm. See also Movie S1.

D. Myo2-4IQ-3GFP transporting RFP-Snc1 positive vesicle to bud. Arrowheads show vesicle/motor puncta colocalization. Scale bar, 2 µm. See also Movie S2.

E. Histogram of number of motors on RFP-Snc1 positive diffusing vesicles, n= 94 puncta.

F. Histogram of number of motors on RFP-Snc1 positive vesicle undergoing active transport, n= 94 puncta.

G. GFP-Sec4 positive diffusing vesicles, as indicated by arrowheads. Scale bar, 2 µm. H. GFP-Sec4 vesicle undergoing active transport, as shown by arrowheads. Scale bar, 2 µm.

I. Histogram of number of GFP-Sec4 molecules per diffusing vesicle, n= 98 puncta.

J. Histogram of number of GFP-Sec4 per puncta undergoing active transport, n = 72 puncta.

K. Number of molecules of Myo2-GFP and GFP-Sec4 in the total bud and bud tip of medium-budded cells, using total molecules per cell information obtained from quantitative immunoblots and whole cell fluorescence projections of cells. Error bars represent standard deviations for n= 55, 39 cells, respectively. See also Figure S2.

Timelapse micrographs of Myo2–3GFP show diffusing puncta in the mother cell that then move abruptly towards the bud. To define the number of motors present on a secretory vesicle in different stages of its delivery cycle, we replaced chromosomal MYO2 with myo2–4IQ-3GFP. This allele contains four IQ motifs rather than the normal six in its lever arm, resulting in a truncated lever arm that reduces secretory vesicle transport speed (2.00 ± 0.56 µm/s, n= 25). Use of the slower transporting mutant was necessary to capture sufficient images of moving vesicles for quantitation, and does not affect protein expression, growth rate or Myo2p localization (Schott et al., 2002). The Myo2–4IQ-3GFP puncta colocalize with RFP-Snc1 puncta, a tagged version of the post-Golgi v-SNARE that resides on secretory vesicles (Figure 2C and Movie S1; Protopopov et al., 1993). Nearly all Myo2–4IQ-3GFP puncta were positive for RFP-Snc1, whereas about half of the RFP-Snc1 puncta were positive for Myo2–4IQ-3GFP. Importantly, all rapidly moving RFP-Snc1 puncta had associated Myo2–4IQ-3GFP (Figure 2D and Movie S2). Thus, secretory vesicles marked by RFP-Snc1 recruit Myo2p and are rapidly transported to growth sites, presumably when they encounter an actin cable.

We next sought to determine the number of motors involved in delivering secretory vesicles to sites of growth through in vivo quantitative microscopy. The number of molecules of Myo2–4IQ-3GFP on RFP-Snc1 vesicle puncta were estimated by coimaging with yeast expressing the centomeric histone protein Cse4-3GFP as a molecular standard, assuming ~80 Cse4-3GFP per anaphase cluster (Lawrimore et al., 2011). We find that there are 8.7 ± 3.2 motors per diffusing secretory vesicle (Figure 2E), and 10.9 ± 4.1 motors on vesicles being actively transported into the bud (Figure 2F). Because all rapidly moving vesicles were positive for Myo2–3GFP, it is unlikely we are under-sampling the number of motors per vesicle.

As Sec4p is part of the receptor for Myo2p on secretory vesicles, we also determined the number of Sec4p on each secretory vesicle. Sec4p is C-terminally prenylated, so we appended GFP to the N-terminus of Sec4p at its chromosomal locus, which caused no deleterious effects. Quantitative immunoblotting indicates there are about 6,900 GFP-Sec4 molecules in a cell (Figure 2B). Confocal microscopy showed randomly diffusing puncta of GFP-Sec4 that, like Myo2p-3GFP, were seen to abruptly move into the bud (Figures 2G and 2H). We estimate about 54 ± 24 molecules of GFP-Sec4 per diffusing particle (Figure 2I), and 75 ± 22 molecules on puncta undergoing active transport to sites of growth (Figure 2J). Thus, there is an excess of Sec4p molecules to accommodate the ~10 Myo2p motors on each puncta even if each Myo2p binds two Sec4p as would be expected.

Do the puncta we see with Myo2–4IQ-3GFP, RFP-Snc1 and GFP-Sec4 correspond to single post-Golgi secretory vesicles, or clusters of them? A 5µm diameter yeast bud requires about 2000 secretory vesicles (~80–100 nm in diameter) to generate sufficient membrane surface. Post-Golgi secretory vesicles have to also provide membrane for endocytosis; given the number of endocytic patches and their lifetimes in the bud, a 30–50 nm endocytic vesicle is generated about every three seconds (Prescianotto-Baschong and Riezman, 1998; Kaksonen et al., 2003). If we assume a doubling time of 90 minutes, cell growth and endocytosis would require the delivery of about one vesicle every 2–3 seconds, which is close to what we observe (0.29 ± 0.13 vesicles moving per second, n=10 movies). This rate implies that we are visualizing and quantifying the components of individual secretory vesicles.

Myo2p remains associated with secretory vesicles upon reaching sites of growth

Vesicles accumulate at sites of growth in the bud due to a kinetic delay before exocytosis; electron micrographs have shown that small growing buds accumulate a cluster of about 10–20 secretory vesicles (Mulholland et al., 1994). To explore the fate of Myo2p after secretory vesicle delivery to sites of growth, we determined the number of Sec4p and Myo2p molecules that accumulate in the bud using our previously obtained quantitative immunoblot data and full cell projections of Myo2-GFP and GFP-Sec4 fluorescence (Figures 2K and S2) About 4260 Sec4p molecules accumulate within a medium sized bud, with 2510 concentrated at the bud tip (n= 39 cells). This tip-localized pool represents Sec4p molecules from approximately 34 vesicles. We also found 1834 molecules of Myo2p (917 motors) in the bud and 828 molecules (414 motors) at the bud tip (n= 55 cells). Strikingly, this represents 38 vesicle complements of Myo2p motors at the bud tip. This implies that a full complement of Myo2p motors remains associated with secretory vesicles until they undergo exocytosis.

Bulk movement of Myo2p motors into the bud is rapid

Since the foregoing analysis represents the steady state view of secretory vesicle transport, we used photobleaching approaches to examine the dynamics of Myo2p. We performed fluorescence recovery after photobleaching (FRAP) experiments on medium-sized buds (2 µm in diameter) of wild-type cells expressing endogenous Myo2-GFP; after bleaching the bud, fluorescence recovery was monitored every second (Figure 3A and Movie S3). This approach therefore monitors a combination of diffusion and active transport into the bud.

Figure 3. Bulk motor and vesicle movement into the bud is rapid.

Figure 3

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A. Still-frame micrographs of FRAP experiment shows rapid restoration of Myo2-GFP polarity. Circle indicates bleach area. Scale bar, 2 µm. See also Movie S3.

B. Normalized Myo2-GFP intensity in the bud following bleach event. Error bars are standard deviation of n= 12 cells. Full recovery not observed since about 33% of all Myo2-GFP is found in bud.

C. Representative still frame micrographs of GFP-Sec4 during FRAP experiment. Circle indicates bleach area. Scale bar, 2 µm. See also Movie S4.

D. Normalized GFP-Sec4 intensity in the bud following bleach event. Error bars are standard deviation of n= 10 cells. Full recovery of GFP-Sec4 not observed because a large fraction of GFP-Sec4 is found in the bud.

We find that Myo2-GFP recovers quickly into the bud, with a Myo2-GFP recovery rate (k) of 0.070 ± 0.014 s−1 (n= 12; Figure 3B). This yields a half-time of recovery (t1/2) of 10.3 ± 1.9 seconds. Similar recovery dynamics were obtained with GFP-Sec4 (Figures 3C and 3D, and Movie S4), with a recovery rate (k) of 0.054 ± 0.007 s−1 and a half-time of recovery of 13.1 ± 1.7 seconds (n= 10). As expected, these values match other vesicle-associated proteins such as exocyst components (Boyd et al., 2004) and indicate that bulk transport of secretory vesicles to sites of growth is rapid.

Myo2p resides in the bud for a defined time and is then deactivated

To estimate the duration Myo2-GFP resides in the bud, we undertook a Fluorescence Loss in Photobleaching (FLIP) analysis of wild-type, medium-budded cells; the mother cell was photobleached every six seconds and the loss of fluorescence from the bud monitored every 2 seconds. Our photobleaching setup allows for precise control over the region photobleached, and performing this experiment in fixed cells showed that the bud is not photobleached in our FLIP experiments (Figure S3a–b). Further, we used an area of the field of view to subtract background and a separate unbleached cell to correct for the small amount of photobleaching that occurs during the observation period.

FLIP analysis revealed that Myo2-GFP fluorescence was lost from the bud in a highly reproducible manner over the course of 90 seconds (Figure 4A and Movie S5). To obtain rates of motor loss from the bud in such a FLIP experiment, we plotted the normalized fluorescence intensity of Myo2-GFP in the bud over time (Figures 4B and S3c). We determined that two components constitute an exponential loss of motors from the bud. A fast rate of motors leaving the bud yielded a Myo2-GFP half-time of loss (t1/2) of 9.6 ± 2.1 sec, representing 57% of the motor population, while a slower rate of motors leaving the bud yielded a half-time of loss of 32.5 ± 13.7 sec, representing 36% of the population (n= 12). Approximately 10% of the total motor population in the bud remained immobile. An analysis of these rates is complicated by the fact that we were unable to continuously bleach the mother cell, so some molecules could leave the bud and re-enter without being bleached. Nevertheless, an examination of individual time points shows that fluorescence is retained longer at the bud tip compared to the bud cytoplasm (Figure 4A and Movie S5). The motors at the bud tip (which our data suggests are still bound to secretory vesicles at sites of growth) therefore correspond to the slower rate of loss while the faster rate corresponds to motors in the bud cytoplasm. These two fractions are also close to our steady-state data as approximately 45% of all motors in the bud reside at the bud tip, which is the sum of the immobile and tip localized populations. A similar analysis of GFP-Sec4 found only one rate contributing to the loss of the Rab protein from the bud during a FLIP experiment, with a half-time of loss of 92.7 ± 39.1 sec (n=10) (Figures 4C and 4D).

Figure 4. Myo2-GFP recycles from the bud faster than Sec4-GFP.

Figure 4

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A. Still-frame micrographs of Myo2-GFP during FLIP experiment. Box indicates region bleached repeatedly. Scale bar, 2 µm. See also Figure S3a–c and Movie S5.

B. Normalized Myo2-GFP intensity in the bud during FLIP experiment. Error bars indicate the standard deviation of n= 12 cells. Double exponential line of best fit shown in gray.

C. Still-frame micrographs of GFP-Sec4 during FLIP experiment. Box indicates region bleached repeatedly. Scale bar, 2 µm.

D. Normalized GFP-Sec4 intensity in the bud during FLIP experiment. Error bars indicate the standard deviation of n= 10 cells.

We next examined the recycling dynamics of the constitutively active Myo2–13-GFP mutant. Consistent with its constitutively active nature, Myo2–13-GFP is hyperpolarized to the bud tip, with about 70% of all motors residing in the bud, compared with only 35% for wild-type cells (Figure 5A). FLIP analysis at the permissive temperature shows that Myo2–13-GFP has a slight recycling defect with the tip-localized population having a half-time of loss of 46.3 ± 22.0 seconds (n= 13). After a 5 minute shift to the restrictive temperature it displays a greater recycling defect, with the tip-localized population having a half-time of loss of 99.5 ± 26.1 seconds (n= 16; Figures 5B and 5C). This tip-localized population also increases significantly from 53% to 66% of the total population, while there was no change in the fraction of motors in the ‘immobile’ population. These results show that a constitutively active motor gets trapped at sites of growth, indicating that wild-type Myo2p must become deactivated when it releases its cargo and then recycles back to the mother cell.

Figure 5. Myo2p must be deactivated for efficient recycling back into the mother cell.

Figure 5

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A. Fraction of Myo2-GFP and Myo2-13-GFP in the bud at 21°C and 35°C (5 min shift). Error bars represent standard deviation.

B. Normalized Myo2-GFP and Myo2-13-GFP intensity in the bud during the FLIP experiment at indicated temperature. Error bars are standard deviation of n= 12 (wildtype), 13 (mutant permissive), and 16 (mutant restrictive) cells, respectively.

C. Still-frame micrographs of Myo2-13-GFP FLIP experiment at 21°C and 35°C. Boxed region indicates region bleached repeatedly. Scale bar, 2 µm.

Myo2p recycling from vesicles requires exocyst function but not membrane fusion

Our data imply that Myo2p motors and secretory vesicles are intimately coupled until secretory vesicles reach sites of growth and that motors must deactivate to recycle efficiently. To dissect the steps necessary for efficient recycling of Myo2p from vesicles at sites of growth, we examined the behavior of Myo2–3GFP in a panel of conditional mutants affecting events at the bud tip. Secretory mutants sec6-4 and sec15-1 are temperature-sensitive alleles of core exocyst complex proteins; at the permissive temperature, secretion is normal but upon shifting to the restrictive temperature, the exocyst complex disassembles and secretory vesicles accumulate in the bud (Figure S4a–d; Salminen and Novick, 1987; Govindan et al., 1995; TerBush and Novick, 1995). Similarly, the sec9-4 and sec9-7 conditional t-SNARE mutants display relatively normal secretion at the permissive temperature; shifting to the restrictive temperature impairs the essential SNARE function in fusion and also causes the accumulation of secretory vesicles together with the exocyst complex (Figure S4a–d; Rossi et al., 1997; Katz et al., 1998). We reasoned that if Myo2p release from secretory vesicles required exocyst tethering and/or membrane fusion, there would be an increase in the amount of Myo2–3GFP found in the bud of the relevant mutant during short shifts to the restrictive temperature due to a buildup of the motor with the vesicles.

Whole cell projection images of live wild-type and mutant cells showed that about 33% of all Myo2–3GFP is present in the bud of medium-budded cells at the permissive temperature of all strains tested. However, there was a small but significant increase in the fraction of Myo2–3GFP in the buds of sec6-4 and sec15-1 exocyst mutants following a shift to the restrictive temperature while there was no significant change in the sec9-4 and sec9-7 t-SNARE mutants (Figures 6A and S5a). There was also a small but significant increase in the amount of GFP-Sec4 in all mutants when shifted to the restrictive temperature as expected (Figure 6B and Figure S5b). Critically, these results imply that recycling of motors is delayed when exocyst tethering is inhibited, but not when SNARE function is inhibited.

Figure 6. Efficient Myo2-GFP recycling from the bud requires exocyst complex tethering, but not SNARE action.

Figure 6

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A. Fraction of Myo2–3GFP in the bud from whole cell projections for indicated strains and conditions. Temperature shifts were conducted for 15 minutes. *, p<0.05 significance. See also Figure S5a.

B. Fraction of GFP-Sec4 in the bud from whole cell projections for indicated strains and conditions. Temperature shifts were conducted for 15 minutes. *, p<0.05 significance. See also Figure S5b.

C. Normalized Myo2-GFP intensity in the bud of sec6-4 cells during a FLIP experiment at 21 °C and 35 °C. Error bars represent standard deviation of n= 10 cells. See also Figures S4 and S5c.

D. Normalized Myo2-GFP intensity in the bud of sec9-4 cells during a FLIP experiment at 21 °C and the restrictive 35 °C temperature. Error bars represent standard deviation of n= 12 cells (permissive), 16 cells (restrictive). See also Figures S4 and S5d.

To address how the recycling dynamics of Myo2p change when we perturb essential exocyst tethering or SNARE functions, we performed FLIP experiments with Myo2-GFP in wild-type, sec6-4, sec15-1, sec9-4 and sec9-7 strains at both the permissive and restrictive temperatures (Figures 5B, 6C, 6D, S5c–d). No significant difference was observed in the rates and half-times of loss of vesicle-bound Myo2-GFP leaving the bud in wild-type cells, or in the sec9-4 or sec9-7 t-SNARE mutants at either temperature. However, in the sec6-4 and sec15-1 exocyst mutants, the vesicle-associated population of Myo2-GFP took 2 to 3-fold longer to leave the bud at the restrictive temperature. This implies that efficient Myo2p recycling from sites of growth requires vesicle tethering to the plasma membrane through the exocyst complex while SNARE function is not required for efficient Myo2p recycling.

Delaying hydrolysis of Sec4-GTP slows Myo2p recycling

We next sought to explore which factors might contribute to efficient recycling of Myo2p motors from sites of growth. The Rab GTPase Sec4p coordinates many aspects of secretory vesicle transport, docking, and fusion, as its known effectors include Myo2p (Santiago-Tirado et al., 2011), the exocyst component Sec15p (Guo et al., 1999), the lgl homologs Sro7/77p (Gangar et al., 2005), and the Sec1p–targeting factor Mso1p (Weber-Boyvat et al., 2011). To explore if Sec4-GTP hydrolysis influences Myo2p recycling from growth sites, we examined the effect of delaying hydrolysis by deleting one or both of the bud-cortex localized, redundant GTPase Activating Proteins (GAPs) for Sec4p, Msb3p/Msb4p (Gao et al., 2003).

In FLIP experiments, no significant difference in the half-times of loss of Myo2-GFP from the bud were found between wild-type and the msb3A or msb4A single GAP mutants (Figures 7A and 7B). However, when both GAP proteins were deleted (msb3A msb4A), Myo2-GFP half-times of loss from bud of the vesicle-associated fraction increased 2 to 3-fold. This increase was not due to an increase in the immobile pool of motors and was concurrent with a build up of GFP-Sec4 and Myo2–3GFP in the bud of the msb3Δ msb4Δ cells (Figures 7C–E); this suggests that delaying GTP hydrolysis results in both an accumulation of secretory vesicles and kinetic delay in motor recycling.

Figure 7. Deletion of Sec4-GAPs Msb4p and Msb4p or using the constitutively active sec4-Q79L mutant significantly delays Myo2-GFP recycling.

Figure 7

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A. Representative summed projection micrographs of Myo2-GFP in bud during FLIP experiment for wild-type, msb3Δ, msb4Δ, and msb3Δ msb4Δ cells. Scale bar, 2 µm.

B. Normalized Myo2-GFP intensity in the bud of wild-type, msb3Δ, msb4Δ, and msb3Δ msb4Δ cells during FLIP experiment. Error bars represent standard deviation of n= 11 cells for each strain.

C. Maximum projection micrographs of Myo2-3GFP in wild-type and msb3Δ msb4Δ cells. Scale bar represents 2 µm.

D. Fraction of Myo2-3GFP in the bud from whole cell projections for wild-type, msb3Δ, msb4Δ, and msb3Δ msb4Δ cells. Error bars represent standard deviation. *, p<0.05 significance.

E. Fraction of GFP-Sec4 in the bud from whole cell projections for wild-type, msb3Δ, msb4Δ, and msb3Δ msb4Δ cells. Error bars represent standard deviation. *, p<0.05 significance.

F. Normalized Myo2-GFP intensity in the bud of wild-type and sec4-Q79L during FLIP experiment. Experiment done following 15 minute shift to the cold sensitive temperature of 14 °C. Error bars represent standard deviation of n= 13 cells for each strain.

G. Model of Myo2p activation, transport, and release from secretory cargo.

To examine the role of Sec4-GTP hydrolysis in another manner, we replaced the chromosomal copy of SEC4 with the sec4-Q79L constitutively active mutant allele in which GTP hydrolysis is delayed. Previous analysis of the sec4-Q79L allele shows that it confers a cold sensitive phenotype, and in vitro Sec4-Q79L has 30% reduced hydrolysis activity compared to wild-type Sec4p (Walworth et al., 1992). FLIP experiments conducted between strains harboring wild-type SEC4 and the sec4-Q79L mutant at the impaired temperature of 14 °C revealed that Myo2-GFP recycling from sites of growth in the bud was delayed similarly to the double msb3Δ msb4Δ GAP deletion mutant (Figure 7F). Thus, when GTP hydrolysis of Sec4p is delayed, exocytosis and the recycling of Myo2-GFP from sites of growth are also delayed.

DISCUSSION

Many studies of class-V myosins have focused on how they interact with their effectors and cargos. Missing from such analyses is the dynamic properties of the motor itself and how its dynamics is regulated at the molecular level. Here we have described the delivery cycle of a class-V myosin in vivo; a model summarizing our results is shown in Figure 7G. Myo2p motors are activated from an inactive pool through binding to a competent secretory vesicle and then the vesicle, in complex with ~10 motors, is shuttled to sites of growth. By quantitating molecules at the bud tip, we find that docked vesicles retain a full complement of motors. Further, vesicle tethering by the exocyst complex and Sec4-GTP hydrolysis are required for efficient Myo2p recycling back into the mother cell, but efficient recycling does not require vesicle fusion. Since the myosin-V family of molecular motors is highly conserved, as are the Rab proteins they interact with and the exocyst complex that regulates their cargo dissociation, our findings are likely to be widely applicable.

Previous data has shown that in myo2 conditional tail mutants shifted to the restrictive temperature, Myo2p polarizes to sites of growth without secretory cargo (Schott et al., 1999). However, wild-type Myo2–3GFP fails to polarize when competent secretory cargo is not available, thereby demonstrating that motors are activated by binding cargo. Importantly, all myo2 tail alleles examined that have conditionally lost their ability to transport secretory vesicles at the restrictive temperature remain polarized (Schott et al., 1999; this study), suggesting that the inability to bind secretory vesicles correlates with Myo2p activation. Consistent with this model, constitutively active Myo2–13p hyperpolarizes to sites of growth and is recycled from the bud incredibly slowly at the restrictive temperature. Thus, wild-type Myo2p needs to be activated by cargo, and deactivated upon cargo delivery.

How is Myo2p activated by secretory vesicles? In vitro studies with mammalian myosin-V have suggested that the motor undergoes autoinhibition through an interaction of the ATPase-containing head domain and the cargo-binding tail domain, and key residues in the tail mediating this regulation have been identified (Liu et al., 2006; Thirumurugan et al., 2006; Li et al., 2008). An attractive model for Myo2p activation is that the autoinhibited motor becomes active by binding to the receptor complex on secretory vesicles. Known components of the secretory vesicle receptor complex that interact with Myo2p include the exocyst component Sec15p (Jin et al., 2011), the Rab Sec4p, and an unknown component dependent on PI4P (Santiago-Tirado et al., 2011). A recent study found that the Sec15p binding site on the Myo2p tail overlaps with the conserved residues believed to be involved in autoinhibition (Jin et al., 2011). However, the observation that secretory vesicles are still transported to sites of growth in the sec6-4 and sec15-1 mutants (Salminen and Novick, 1987; Govindan et al., 1995), where the exocyst is disassembled and Sec15p is depolarized (Terbush et al. 1995; this study), makes it unlikely that Sec15p is critical for Myo2p activation. A more likely regulator of the activation process is the Rab Sec4p, especially as we have found that Myo2p deactivation is slowed in mutants where Sec4-GTP hydrolysis is delayed. Moreover, Myo2p is depolarized and therefore not activated in sec4-8 mutant cells at the restrictive temperature, suggesting that Sec4p binding affects the regulation of Myo2p. It is presently not known if Myo2p undergoes a head-tail interaction or what the physiological significance of such an interaction would be.

The association of ~10 motors with a transporting secretory vesicle raises the question of how many motors are needed and why there are so many. Their abundance would solve a longstanding question first described with in vitro work, which suggest that Myo2p is not nearly as processive as its mammalian MyoV homologs (Reck-Peterson et al., 2001). It has been suggested that at least 5 motors would need to be present on a cargo in order to obtain processive motion (Reck-Peterson et al., 2001), so our findings are consistent with this reasoning. However, we also found that Myo2p defective in binding cargo becomes constitutively active as indicated by its highly polarized accumulation and through FRAP experiments in the absence of cargo. This implies that even in the absence of multimerization by binding a secretory vesicle, Myo2p can move processively down actin filaments. While this paper was in the final stages of assembly, Hodges et al. (2012) reported that in vitro Myo2p becomes a processive motor with the addition of tropomyosin. This observation makes it likely that long run-lengths along actin cables can be achieved in the cell, but perhaps several motors are needed to allow for long-range transport of secretory cargo.

We find about 9 myosin-V motors on vesicles freely diffusing around the cytoplasm. This suggests that the rate-limiting step for transport is engaging an actin cable in the cytoplasm. Further, the normal distribution of the number of motors on this class of vesicles suggests that motors must be acquired rapidly; if they were acquired slowly one would expect to see more vesicles with smaller numbers of motors. Since we also find 75 molecules of Sec4p per transported vesicle, there are more than enough for the two Myo2p tail domains to bind, as might be expected.

When secretory vesicles arrive at sites of growth, they accumulate there because exocytosis is a kinetically slower process than transport. The docked vesicles, as marked by Sec4p, retain a full complement of Myo2p; this suggests that exocytosis is coupled with motor release and deactivation. This model derives from the observation that a constitutively active motor resides at the bud tip much longer than wild-type Myo2. We next investigated how Myo2p might be deactivated and released. Motor recycling back into the mother cell was significantly delayed when tethering by the exocyst complex was disrupted. Further, there was an increase in total Myo2p and Sec4p found in the bud of these mutants, consistent with vesicle accumulation and maintenance of the Sec4-GTP/Myo2p interaction. Analysis of t-SNARE sec9 conditional mutants did not show a kinetic delay in the recycling of Myo2p, nor was there a buildup of total Myo2p in the bud compared to the non-permissive condition. Critically, these results show that efficient Myo2p recycling requires exocyst complex tethering but is independent of the membrane fusion step requiring Sec9p. Further experiments demonstrate that delay in Sec4-GTP hydrolysis by deleting the redundant Sec4p GAP proteins Msb3/4p or using the constitutively active sec4-Q79L mutant also slows motor recycling, indicating that motor deactivation is related to Sec4-GTP hydrolysis.

The interpretation of these findings is not straightforward as there are at least four effectors of Sec4-GTP in the bud tip: Myo2p (Santiago-Tirado et al., 2011), the exocyst component Sec15p (Guo et al., 1999), the Sec1p–tartgeting factor Mso1p (Weber-Boyvat et al., 2011), and the Lgl homolog Sro7/77p that interacts with Sec9p and is involved in v-/t-SNARE fusion (Gangar et al., 2005). The functional relationships between these different effectors are not yet known, especially if there is a temporal order in which Sec4-GTP interacts with its various effectors at the bud tip. Nevertheless, our results clearly show that motor recycling is influenced by the presence of Sec4-GTP and coordinated with events at the bud tip.

With this data, we can propose a framework for how the delivery cycle of Myo2p is coordinated with events at sites of growth. First, Myo2p delivers vesicles to the bud tip through its interaction with Sec4-GTP, and a component dependent on PI4P. Myo2p is also integrated with the vesicle-associated exocyst complex through its interaction with Sec15p (Jin et al., 2011). Second, the vesicle-transported and cortex-localized components of the exocyst tether the vesicle, together with Myo2p, at the cortex. The presence of Sec4-GTP and/or Myo2p stimulates Sro7/77p to displace Sec9p, priming the interaction between Sso1p and the v-SNARE Snc1/2p. Next, Sec4-GTP hydrolysis is activated by its GAP, Msb3/4p, resulting in release of Myo2p and Sec15p from Sec4p and eventual downstream fusion. An attractive possibility for coordinating this process would be if the assembled exocyst complex stimulates the GAP activity of Msb3/4p. It is interesting to note that defects in exocytosis result in the accumulation of hundreds of secretory vesicles as seen by electron microscopy, yet we only see about a 1.4-fold accumulation of Sec4 and Myo2 in conditional exocyst mutants. This implies that vesicles have a window of opportunity to fuse, and if they miss it, they either have to recruit fresh Sec4-GTP, or never fuse. How timing of events at the bud tip is determined will be a critical issue to be defined in future studies, but our data provides an outline for how Myo2p release is coordinated with events at sites of growth.

Post-Golgi secretory trafficking proteins are well conserved in higher eukaryotes, and it is likely that a closely related mechanism for activation and release of Myo2p seen in yeast exists for myosin-Vs in mammalian cells. The tail domain of myosin-Va was recently found to interact with the Sec4p homolog Rab3a, which resides on synaptic vesicles in neurons (Wöllert et al., 2011). This interaction allows for the mobilization of AMPA receptors after long-term potentiation and proper activation and release of the motor would be of obvious importance. Similarly, Glut4-positive vesicles are transported to the plasma membrane in muscle cells upon insulin stimulation through the interaction of myosin-Vb and the close Sec4p homolog Rab8a (Ishikura and Klip, 2008). Since the exocyst is likely linked to Rab8 through its interaction with Sec15 (Wu, S. et al., 2005), the same basic framework of tethering and release of the motor through Rab-GTP hydrolysis is likely to apply here as well. Further studies in mammalian cells may show conservation of motor activation by competent secretory vesicles and release following tethering and Rab-GTP hydrolysis.

EXPERIMENTAL PROCEDURES

Yeast Strains and Molecular Biology Techniques

Strains used in this study are listed in Supplemental Experimental Methods. Cells were grown using standard laboratory techniques (Sherman, 2001). Yeast transformations were performed using lithium acetate methods (Gietz et al., 2002). Chromosomal GFP tagging performed as described in Supplemental Experimental Procedures. Actin patches and filaments were visualized using phalloidin staining (Liu et al., 2012).

Microscopy and Photobleaching

Micrographs were acquired with a CSU-X spinning disc confocal microscope system (Intelligent Imaging Innovations) using a DMI600B microscope (Leica) and QuantEM EMCCD camera (Photometrics) controlled by Slidebook 5.0 Software (Intelligent Imaging Innovations). Strains shifted to high temperatures were imaged in an environmental chamber (Okolab) at the indicated temperature. Strains were imaged for short experiments on a 1.5% QSD agarose pad (Rossanese et al., 2001); for longer experiments or when photobleaching, a glass-bottomed dish with 0.5 mg/mL Concanavalin A (EY Laboratories) pre-spotted to adhere cells to the glass was used. Images were processed in either ImageJ or Slidebook 5.0 software. Panels were assembled after identical processing unless otherwise indicated.

Quantification of molecule numbers on secretory vesicles was done largely by following established protocols (Joglekar et al., 2008). The new assumption of ~5 molecules of Cse4p per kinetochore, or ~80 per anaphase cluster (Lawrimore et al., 2011) was used. An RFP-SNC1 CEN plasmid acquired from Ruth Collins at Cornell University was used to determine if Myo2–4IQ-3GFP was on secretory vesicles. This was coimaged with a Cse4-3GFP strain (kindly provided by Dr. Wei-Lih Lee, University of Massachusetts, Amherst) for direct comparison of fluorescence intensities. Similarly, a Cse4-GFP strain was directly compared to GFP-Sec4 strain. All cells were grown to log phase in synthetic media. Comparison of Cse4p foci intensity (with nuclear background subtracted) of cells in anaphase and vesicle foci intensity (with cytoplasm background subtracted) allowed for the determination of molecule number on vesicles. Several single plane movies centered on the bud neck were acquired for each strain. Vesicles diffusing around the cytoplasm were defined as those vesicles that don’t make bud-directed movements over five frames (~2.2 seconds); the brightest intensity was used to determine molecule number per diffusing vesicle. Vesicles undergoing active transport to sites of growth were defined as vesicles that make rapid bud-directed movement over three frames; the brightest intensity was used to determine molecule number per vesicle.

Photobleaching experiments were performed using an argon laser and mosaic digital illumination system (Andor Scientific). Medium budded cells (defined as a bud diameter of 2 µm) were used throughout all photobleaching experiments for standardization purposes. FRAP experiments were conducted by photobleaching the central plane of the confocal z-section containing the bud of the bud for 2000 ms and determining the recovery kinetics of the GFP tagged protein for every second thereafter. In FLIP experiments, nearly the entire mother cell was photobleached for 750 ms every 6 seconds; micrographs and the intensity of GFP in the bud was determined every 2 seconds. FLIP experiments in temperature-sensitive strains were shifted to 35 °C for 1 hour unless otherwise indicated.

In all photobleaching experiments, the pre-bleach intensity of tagged protein in the bud was normalized to 1.0 to compare between cells. Further, the photobleaching caused by imaging was normalized using a nearby cell. The number of modes of action contributing to the recovery of signal in a FRAP experiment was found using methods described in Boyd et al. (2004). The half-times of recovery were calculated using the equation t1/2= (ln 2)/k (Salmon et al., 1984). To determine the modes of action in a FLIP experiment, the normalized intensity in the bud on an exponential scale was plotted against time. The presence of two discontinuous slopes was evidence for two rates constituting loss from the bud (Figure S3c). KaleidaGraph (Synergy Software) was used to generate single or double exponential curves to determine rates of recovery or loss. Rates for FLIP and FRAP experiments are summarized in Table S1.

Whole cell projections of log phase, medium budded cells were used to determine the fraction of tagged Myo2p or Sec4p found in the bud. Cells were imaged for 50 ms in each plane of a z-stack (0.5 µm step size), which was then used to create a summed projection for analysis of fluorescence in different cell regions. Background subtraction and quantification was performed in ImageJ. FLIP experiments in Figure 7F were conducted at 14 °C using a peristaltic pump (Rainin Instrument Co Inc.) that moved chilled media across the cells on the glass-bottomed dish. Cells were cooled to 14 °C for 15 minutes prior to conducting FLIP experiment. A temperature probe was used to ensure the temperature consistently remained at the cooled temperature.

Quantitative Immunoblots and Calculation of Molecule Number

Quantitative immunoblots of Myo2-GFP and GFP-Sec4 were performed using purified recombinant GST-eGFP and cell lysates containing chromosomally tagged proteins. To obtain yeast cell lysates, tagged GFP-Sec4 and Myo2-GFP strains were grown to log phase in SD media at 26°C in 8 mL cultures. A hemocytometer was used to determine the number of cells per culture. Cells were resuspended in 70 µL disruption buffer (20 mM Tris-Cl pH 7.9, 10 mM MgCl2 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 0.3 M ammonium sulfate, 1 mM PMSF, 1X Sigma yeast protease inhibitor cocktail) with 0.1 g acid-washed glass beads (Sigma). 6 × 1 minute vortex cycles at 8 °C (with one minute on ice between cycles) was used to disrupt cells. Empirical tests determined that nearly all cells were disrupted after six cycles. Sample buffer was then added directly, boiled for 1 minute, and clarified by centrifugation to obtain a crude lysate. Three independent preparations per protein were done when calculating number of molecules per cell.

To determine the number of proteins per cell, quantitative immunoblots were performed and analyzed using Odyssey infrared imaging system (LI-COR Biosciences). Essentially all proteins were transferred after a 1.5 hour semi-dry transfer. Mouse monoclonal antibody against GFP (Santa Cruz Biotechnology) was used to probe the membrane and the amount of GFP-tagged protein per cell was determined.

To obtain the number of molecules in different areas of the cell, whole cell z-projections of Myo2-GFP and GFP-Sec4 strains were obtained by confocal microscope using a 100X objective, 2X photomultiplier, and 0.4 µm step-size. We assumed that the fluorescence intensity in the yeast cell is directly comparable to the number of molecules present (Wu, J-Q. and Pollard, 2005). Strains were grown in the dark to log phase at room temperature. Images were processed in ImageJ; after background subtraction, the intensity of signal in the mother, bud, and bud tip was then related to the number of proteins per cell obtained by quantitative immunoblot to determine molecule number in the mother, whole bud, and bud tip regions.

Statistical Methods

Student’s t-test was used to determine significance between samples. A 95% confidence interval (p<0.05) was determined to be significant.

Supplementary Material

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Research Highlights for Website.

  • Myo2 is activated by transport-competent secretory vesicles

  • 10 motors remain associated with secretory vesicles until reaching the bud cortex

  • Efficient motor recycling requires the exocyst complex but not SNARE action

  • Rab-GTP hydrolysis regulates Myo2 recycling from sites of exocytosis

ACKNOWLEDGEMENTS

We are grateful to members of the Bretscher lab for discussion and reading this manuscript. We also thank P. Brennwald, E. Bi, P. Novick, R. Schekman, and W.-L. Lee for providing strains used in this study. This work was supported by NIH grant GM39066.

Footnotes

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