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Yeast Eps15-like endocytic protein Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton

  1. Junko Y Toshima
  2. Eri Furuya
  3. Makoto Nagano
  4. Chisa Kanno
  5. Yuta Sakamoto
  6. Masashi Ebihara
  7. Daria Elisabeth Siekhaus
  8. Jiro Toshima  Is a corresponding author
  1. Tokyo University of Technology, Japan
  2. Tokyo University of Science, Japan
  3. Institute of Science and Technology Austria, Austria
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Cite this article as: eLife 2016;5:e10276 doi: 10.7554/eLife.10276

Abstract

The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles. In yeast, endocytic vesicles move towards early endosomes along actin cables, however, the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood. The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases. Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines, pan1-18TA, displayed almost the same endocytic defect as ark1Δ prk1Δ cells, and contained abnormal actin concentrations including several endocytic compartments. Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables. The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables, and between endocytic vesicles and endosomes. Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables.

https://doi.org/10.7554/eLife.10276.001

eLife digest

The cells of all eukaryotes – including plants, animals and fungi – absorb many substances that they need from their surroundings by forming pockets around them, and then pinching off these pockets to create structures called vesicles. Clathrin is a protein that acts as a scaffold for these vesicles.

Inside a eukaryotic cell, clathrin-coated vesicles first go to a structure known as an endosome, possibly by following a track made from filaments of a protein called actin. Researchers have shown previously that a yeast protein called Pan1 binds to actin filaments and helps the cells to create clathrin-coated vesicles. However it was unclear if the Pan1 protein is also important for transporting clathrin-coated vesicles to endosomes.

Previous studies have also shown that adding phosphate groups on to the Pan1 protein prevents it from binding to clathrin-coated vesicles or actin filaments. Now, Toshima et al. show that a mutant version of the Pan1 protein, which cannot be modified in this way, can bind stably to both clathrin-coated vesicles and the actin filaments and connect them together. The experiments also showed that, in yeast cells that only produce the mutant version of Pan1, clathrin-coated vesicles bind stably to endosomes without the need for actin. Thus, these findings show that the addition of phosphate groups onto Pan1 is part of a mechanism that regulates the interactions between clathrin-coated vesicles, endosomes and actin filaments.

Following on from this work, one future challenge is to find which proteins directly connect clathrin-coated vesicles with endosomes. It will also be important to investigate if similar mechanisms are used in the cells of mammals.

https://doi.org/10.7554/eLife.10276.002

Introduction

Endocytosis is the process by which cells internalize various molecules, such as proteins and lipids, from the plasma membrane and outside the cell. Recent live-cell imaging studies of yeast and mammalian cells have revealed that the actin cytoskeleton plays important roles in the formation and internalization of clathrin-coated vesicles (CCVs) and post-internalization events in the endocytic pathway, including vesicle transport and endosome motility (Engqvist-Goldstein and Drubin, 2003; Girao et al., 2008; Kaksonen et al., 2006). In the early stages of the endocytic pathway, transient actin polymerization at the endocytic site is required for the formation and internalization of a CCV (Kaksonen et al., 2003; Merrifield et al., 2002). This process is regulated by several actin nucleation promoting factors (NPFs), including the type I myosins Myo3/5p, the actin binding protein Abp1p, the yeast WASP homologue Las17p, and the Eps15-like protein Pan1p (Goode et al., 2015; Weinberg and Drubin, 2012). Compared to Las17p and Myo3/5p, Pan1p has a lower NPF activity, and mutation in the Arp2/3 complex binding region of Pan1p causes only a minor defect in actin polymerization at endocytic sites because of the functional redundancy with Las17p (Sun et al., 2006; Toshima et al., 2005). However, a form of Pan1p mutated to prevent phosphorylation at 15 threonines, Pan1-15TA, directly binds to F-actin with high affinity and its expression causes abnormal cytoplasmic actin concentrations, also called actin clumps (Toshima et al., 2005). Thus, Pan1p could be acting after F-actin is assembled by other NPFs as well as or instead of during the initiation of actin polymerization during endocytosis.

Pan1p’s abilities to bind F-actin and promote actin polymerization are regulated by the Prk1 family protein kinases Prk1p and Ark1p, which are related to the mammalian proteins GAK and AAK1 (Smythe and Ayscough, 2003). The Prk1 family kinases are important regulators of endocytosis and the actin cytoskeleton in both yeast and mammalian cells (Smythe and Ayscough, 2003). In budding yeast, Ark1p and Prk1p are recruited to endocytic sites 1–2 s after commencement of actin assembly and CCV internalization, and phosphorylate several endocytic proteins, including Sla1p, Ent1/2p, Yap1801/2p, Scd5p, and Pan1p, to disassemble endocytic coat proteins and actin (Cope et al., 1999; Henry et al., 2003; Toret et al., 2008; Watson et al., 2001; Zeng and Cai, 1999; Zeng et al., 2001). Pan1p is one of the key targets of Ark1/Prk1 kinases, and phosphorylation of Pan1p by Ark1/Prk1 kinases is believed to be important for disassembly of the Pan1p complex, composed of several endocytic proteins (Toshima et al., 2005; Wendland and Emr, 1998; Zeng and Cai, 1999; Zeng et al., 2001). Interestingly, disruption of the normal phosphorylation cycle by deletion or chemical inhibition of Ark1/Prk1 kinases leads to the concentration of actin in association with endocytic vesicles (Sekiya-Kawasaki et al., 2003; Toshima et al., 2005), suggesting a role for Pan1p and other substrates in regulating interaction between endocytic vesicles and the actin cytoskeleton.

After being internalized, endocytic vesicles move away from the plasma membrane in an association with actin cables that is still mechanistically unexplained (Huckaba et al., 2004; Toshima et al., 2006). Yeast actin cables, which are bundles of actin filaments that align along the long axis of budding yeast, are crucial for the establishment of cell polarity (Yang and Pon, 2002). Actin cables are also used as tracks for polarized transport during the secretion of exocytic vesicles and the segregation of organelles from mother to daughter cells (Bretscher, 2003). Many of these types of transport along actin cables are known to depend on the type V myosins, Myo2/4p, which mediate the movement of cargo from the minus to plus ends of actin filaments (Bretscher, 2003). However, transport of endocytic vesicles along actin cables is not likely to depend on these myosins, because a temperature sensitive mutant of MYO2 (myo2-66) or a deletion of MYO4 gene did not exhibit any defect in endocytosis (Govindan et al., 1995; Haarer et al., 1994). Other myosins, such as type II myosin (Myo1p) and type I myosin (Myo3/5p) also do not seem to mediate this transport. Myo1p has an important role in controlling actin cable dynamics at the bud sites or neck, but it is not localized to endocytic vesicles (Huckaba et al., 2006). Myo3/5p are necessary for promoting actin assembly and endocytosis at cortical patches, but they stay at the cell cortex when endocytic vesicles are internalized along actin cables (Sun et al., 2006). Interestingly, a previous study demonstrated that endocytic vesicle movement occurs at the same velocity and in the same direction as the movement of actin cables (Huckaba et al., 2004). They also reported that an endocytic vesicle stays at the same position on the cable and moves together with the actin cable, suggesting that endocytic vesicles are fixed on the actin cables and move as a result of actin cable flow (Huckaba et al., 2004). In addition to endocytic vesicles, early endosomes also associate with the actin cytoskeleton, and the motility of endosomes is significantly inhibited by treatment of latrunculin A (LatA), a drug that sequesters actin monomers (Chang et al., 2003; Fernandez-Borja et al., 2005; Toshima et al., 2006; Voigt et al., 2005). Similarly to endocytic vesicles, early endosome motility also does not depend on Myo2/4p (Toshima et al., 2006). These results suggest that unknown molecular mechanisms exist that bind endocytic vesicles and endosomes to actin cables.

We sought to understand the role of Pan1 phosphorylation during endocytosis using a form of Pan1 that mimics the ark1Δ prk1Δ phenotype. We examined cells expressing Pan1-18TA, which is mutated to prevent phosphorylation at all 18 threonines; this mutant showed almost the same endocytic defect as ark1Δ prk1Δ cells, resulting in stable association between endocytic vesicles and actin cables. Interestingly, the pan1-18TA mutant also leads to accumulation of early endosomes in actin clumps. Thus, phosphorylation of Pan1p seems to regulate the interaction between endocytic compartments and the actin cytoskeleton.

Results

Pan1p is the major in vivo target of Ark1/Prk1 kinases during their regulation of endocytosis

Our group had previously demonstrated that expression of a form of Pan1 containing a mutation of 15 Ark1p/Prk1p consensus sequences (LxxQxTG) to alanine causes an endocytic defect and abnormal clumping of actin in the cytosol. However, the defect in the pan1-15TA mutant was not as pronounced as that in the ark1prk1△ mutant (Toshima et al., 2005). We first sought to determine if the presence of other functionally important phosphorylation sites in Pan1p was responsible for the difference in phenotypes. In a previous intensive investigation, Cai and colleagues identified the [L/I/V/M]xx[Q/N/T/S]xTG motif as a further potential site of phosphorylation by Ark1/Prk1 kinases (Huang et al., 2003). Pan1p contains three more such Ark1p/Prk1p consensus sequences (MQPNIT464G, MMPQTT480G, and MMPQTT487G) all located in the second LR region (Figure 1A) (Huang et al., 2003). When we additionally mutated these sites to create pan1-18TA, we observed a more severe growth retardation phenotype than in pan1-15TA (Figure 1B). The Pan1-18TA protein was expressed normally, but its phosphorylation was mostly inhibited (Figure 1C). pan1-18TA mutant cells displayed prominent actin concentrations and a more severe defect in endocytic internalization (Figure 1D,E). Pan1-18TA-GFP also showed defects in localization, with 95% colocalizing with actin clumps or smaller, peripheral actin patches, similar to Pan1-15TA (Figure 1F) (Toshima et al., 2005). This is in contrast to wild-type cells, in which Pan1p is recruited to cortical patches early, arriving ~20 s before actin is detected, and associates with actin for ~10–15 s (Kaksonen, 2003), resulting in ~30% of Pan1p colocalizing with Abp1p (Figure 1F).

Figure 1 with 1 supplement see all
Construction and characterization of a Pan1p phosphorylation-site mutant.

(A) Structure of a Pan1p phosphorylation mutant. The two amino-terminal Eps15 homology (EH) domains, long repeat (LR) regions, predicted coiled-coil (CC), acidic region (A), and carboxy-terminal prolin-rich domain (PR) domain, are indicated. The fifteen consensus phosphorylation sites previously mutated in Pan1-15TA are indicated below the protein in black. The three additional sites mutated in Pan1-18TA are in red. (B) Plate showing the growth phenotype of pan1-15TA and pan1-18TA mutants. A dilution series of cells was plated on YPD plates and incubated for 2–3 days at 25 or 37°C, respectively. (C) Analysis of the phosphorylation state of the Pan1-18TA mutant. Protein expression was analyzed by immunoblotting 20 μg of total cell lysate (TCL) with an anti-GFP antibody (left panel). Phosphorylated proteins were purified from TCL with Phos-tag agarose, run on SDS-PAGE and immunoblotted with the anti-GFP antibody (right panel) as described in Materials and methods. Lane 1, JJTY369; lane 2, JJTY509; lane 3, JJTY5486. (D) Alexa Fluor 488-phalloidin staining of fixed wild-type and pan1-18TA cells to visualize actin. (E) The effect of Pan1p phosphorylation-site mutations on endocytic internalization. Radiolabeled α-factor internalization assays performed on wild-type (blue), pan1-15TA (yellow), pan1-18TA (magenta), pan1-18TA sla1-10TA (green), or ark1prk1△ (black) cells at 25°C. Each curve represents the average of three independent experiments, and error bars indicate the SD at each time point. (F) The localization of Pan1-GFP in wild-type and pan1-18TA cells. Cells expressing Pan1-GFP and Abp1-mCherry were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy. Merged images of GFP and mCherry channels are shown in the right panels. (G) Endocytic cargo is transported to the vacuole through the actin clumps in pan1-18TA. pan1-18TA cells were labeled with A594-α-factor as described in the Methods. The images were acquired simultaneously at 1, 5, 15, and 30 min after washing out unbound A594-α-factor and warming the cells to 25°C. Scale bars, 2.5 μm.

https://doi.org/10.7554/eLife.10276.003

We next entertained the hypothesis that Prk1p phosphorylation of some of its other known targets such as Sla1p, Ent1/2p, Yap1801/2p and Scd5p (Watson et al., 2001; Zeng et al., 2001; 2007), might also play a role in regulating actin organization and endocytosis. These target proteins were shown to be phosphorylated by Prk1p in vitro, but significant phenotypes caused by mutations of their phosphorylation-sites have not been observed (Henry et al., 2003; Huang et al., 2003; Watson et al., 2001; Zeng et al., 2001). Sla1p contains the most potential Prk1 phosphorylation sites among these proteins (Zeng et al., 2001). We therefore mutated the threonines in all 10 of these [L/I/V/M]xx[Q/N/T/S]xTG sites (Huang et al., 2003) to alanine (sla1-10TA), integrated this mutant into the endogenous SLA1 locus, and analyzed the phenotypes (Figure 1—figure supplement 1A). We first confirmed that the Sla1-10TA mutant was expressed at similar levels to the wild-type protein, and that its phosphorylation was severely inhibited (Figure 1—figure supplement 1B). While cells lacking the SLA1 gene were temperature-sensitive for growth at 37°C, the sla1-10TA mutant exhibited almost the same growth as wild-type cells (Figure 1—figure supplement 1C). Alexa Fluor 488-phalloidin staining of F-actin in fixed sla1-10TA mutants closely resembled that of wild-type cells, with brightly stained actin patches and weakly stained actin cables (Figure 1—figure supplement 1D). We next examined the dynamics of the clathrin-coat module and the actin patches in the sla1-10TA mutants, using GFP-tagged Sla1-10TA and Abp1-mCherry as markers respectively (Kaksonen et al., 2003). Consistent with previous reports, Sla1p and Abp1p patches formed in the cell cortex with lifetimes of 36 ± 7 s and 13 ± 3 s, respectively, culminating in inward movement (Figure 1—figure supplement 1E,F) (Kaksonen et al., 2003). Sla1-GFP localization was immediately followed by a burst of Abp1-mCherry recruitment in wild-type cells (Figure 1—figure supplement 1E). In the sla1-10TA mutants, Sla1p and Abp1p patches formed and disappeared with the typical inward movement, and their lifetimes were slightly prolonged to 43 ± 11 s and 15 ± 3 s, respectively (Figure 1—figure supplement 1F). We also examined the effect of sla1-10TA mutants on endocytic internalization by assessing the ingression of 35S-labeled α-factor, and found that it was only slightly affected (Figure 1—figure supplement 1G). Furthermore, the pan1-18TA sla1-10TA double mutant exhibited only a negligible additive effect, when compared to the pan1-18TA single mutant (Figure 1E, and Figure 1—figure supplement 1D,H). These findings indicate that Pan1p is the major in vivosubstrate of Ark1/Prk1 kinases during their regulation of endocytosis.

We next visualized sequential steps in the endocytic pathway in the pan1-18TA mutant using Alexa Fluor 594-labeled yeast mating pheromone α-factor (A594-α-factor), a marker of endocytosis (Toshima et al., 2006). Interestingly, internalized A594-α-factor moved to actin clumps in pan1-18TA before being transported to the vacuole, while the actin remained in clumps (Figure 1G). This result suggests that the endocytic cargo can transit through the actin clumps before arriving at the vacuole.

Early endosomes are localized at actin clumps in the pan1-18TA mutant

We next sought to determine if other organelles along the endocytic route also accumulate in these actin clumps. To this end, we employed Vps21p and Sec4p as markers of Rab proteins that function in the endocytic or exocytic pathway (Hutagalung and Novick, 2011), Vps8p and Vps11p as markers of the CORVET and HOPS complexes (Balderhaar and Ungermann, 2013), a set of proteins from the ESCRT complex (Hse1p, Mvb12p, Vps36p, and Vps24p) (Bilodeau et al., 2002; Hurley, 2008), Vps4p, Ear1p, and Vps15p as markers of MVBs (Ear1p, and Vps15p) (Burda et al., 2002; Leon et al., 2008), Vps26p as a marker of the retromer complex (Seaman, 2004), Vps52p as a marker of the GARP (Golgi-associated retrograde protein) complex (Bonifacino and Hierro, 2011), and Sec7p as a marker of the trans-Golgi network (Franzusoff et al., 1991). None of these proteins show clump-like localization in wild-type cells (Figure 2—figure supplement 1). Among the 14 proteins examined, three – Hse1p, Mvb12p, and Vps36p – showed a clear change in localization to Abp1-mCherry-labeled actin clumps in the pan1-18TA mutant (Figure 2A,B). Hse1p, Mvb12p, and Vps36p, which function at an early stage of the ESCRT pathway on the way to the MVB, were contained in 60–65% of the actin clumps, whereas Vps24p and Vps4p, which function at a later stage of the ESCRT pathway, were contained in 25–30% of the actin clumps (Figure 2A,B). Vps8p and Vps11p, which mediate early to late transitions of endosomes, exhibited levels of actin clump localization (~30%) similar to that of Vps24p (Figure 2B). In contrast, Vps26p or Vps52p, both of which are required for retrograde transport from late endosomes to the Golgi, showed lower localization (~15% and ~7%, respectively), and Sec7p and Sec4p, which reside on the Golgi or secretory pathway, were rarely contained in actin clumps in the pan1-18TA mutant (<10%) (Figure 2A,B). These results indicate that earlier stage endosomes are highly localized to actin clumps in pan1-18TA mutant cells.

Figure 2 with 1 supplement see all
The localization of endosomal proteins in pan1-18TA cells.

(A) Localization of GFP-tagged endosomal proteins in pan1-18TA. pan1-18TA cells expressing Abp1-mCherry and GFP-tagged endosomal proteins were grown to early to mid-logarithmic phase in YPD medium at 25°C and observed by fluorescence microscopy. Merged images of GFP and mCherry channels are shown in the lower panel. Arrowheads indicate examples of colocalization. Scale bar, 2.5 μm. (B) Quantification of actin clumps including GFP-tagged endosomal proteins. The percentages were calculated as the ratio of actin clumps (= 100) colocalizing with each protein in each experiment. Error bars indicate the standard deviation (SD) from at least three independent experiments.

https://doi.org/10.7554/eLife.10276.005

We wished to confirm that early endosomes accumulate at actin clumps in pan1-18TA mutant cells. As we had seen the ESCRT-0 component Hse1p localize to actin clumps in the mutant, we wished to investigate its organellar localization in wild-type cells more precisely; therefore we tagged Hse1p with three tandem repeats of GFP (3GFP) or mCherry and confirmed their functionality (Figure 3—figure supplement 1A). Hse1-3GFP was clearly detected as numerous small puncta throughout the cytoplasm and prevacuolar compartments (PVCs) (Figure 3—figure supplement 1B). Examining Hse1-3GFP in the overlay of 30 consecutive time-lapse frames made it easy to distinguish the Hse1p localizing at endosomes in the cytoplasm (Figure 3—figure supplement 1B, right cell) and at the PVCs (Figure 3—figure supplement 1B, left cell). By comparing the localization of Hse1-mCherry with GFP-tagged markers, we found that Hse1p exhibited high colocalization with Mvb12p, partial colocalization with Ear1p and Vps26p, and little colocalization with Sec7p (Figure 3—figure supplement 1C,D). We next utilized A594-α-factor to compare the spatiotemporal localization of Hse1p with Vps26p in the endocytic pathway (Figure 3—figure supplement 1E,F) (Toshima et al., 2006; 2009). Hse1-3GFP was highly colocalized with A594-α-factor-labeled endosomes at 5–10 min after α-factor internalization, whereas Vps26-GFP was mainly colocalized with A594-α-factor-labeled endosomes at 10 min (Figure 3—figure supplement 1G). In pan1-18TA cells, similar levels of Hse1p also colocalized with Vps26p (Figure 3—figure supplement 1D,H). These data indicate that Hse1p is widely localized from early to late endosomes and partially colocalized with Vps26p at late endosomes both in wild-type and pan1-18TA cells. Unexpectedly, Vps21p, yeast Rab5, which is known to be localized at early-to-late endosomes (Cabrera et al., 2013; Puchner et al., 2013; Toshima et al., 2014), exhibited lower localization at the actin clumps (~33%), compared to Hse1p (Figure 2A,B). To determine the relative localization of Hse1p and Vps21p at early stage of endocytosis, we slowed endocytic transport by removing glucose from culture medium (Aoh et al., 2011), and compared their localization with internalized A594-α-factor. Consistent with a recent study by Arlt et al. reporting that an ESCRT-I subunit, Vps23, is recruited to endosomes earlier than Vps21p (Arlt et al., 2015), we found that Hse1p colocalizes with A594-α-factor slightly more than Vps21p at 10 min after α-factor internalization (Figure 3—figure supplement 2A). These results, therefore, suggest that endosomes at the early stage of endocytosis are highly localized to actin clumps.

Actin-dependent motility of Hse1p-residing endosomes

The localization of early endosomal proteins at actin clumps in the pan1-18TA mutant suggests that early endosomes might associate with the actin cytoskeleton in wild-type cells. Previous studies have also indicated that actin cables mediate the directed movements of early endosomes (Chang et al., 2003; Toshima et al., 2006), but which endosomes, and how they associate with actin cables, has not yet been clarified. We utilized Hse1p as a marker to address these questions. However because Hse1p is found at early to late endosomes, we classified these endosomes into two categories using Vps26-mCherry: endosomes not labeled with Vps26-mCherry (early stage endosomes) and endosomes labeled with Vps26-mCherry (late stage endosome) (Figure 3A,C). Vps26-mCherry mostly colocalized with Hse1p at the vacuolar membrane (Figure 3A,C). Quantification of endosome velocity revealed that early-stage endosomes moved with an average speed of 125 ± 119 nm/s (= 100), whereas late-stage endosomes moved with an average speed of 156 ± 130 nm/s (= 100) in wild-type cells (Figure 3A,B). We then investigated the effects of the actin-sequestering drug, LatrunculinA (LatA), on the movement of these endosomes. Concomitantly, LatA treatment led to a significant decrease in the velocity of early-stage endosomes (~23 ± 47 nm/s) (Figure 3C,D). In contrast, the velocity of late-stage endosomes was not significantly affected by LatA treatment (~119 ± 112 nm/s) (Figure 3C,D). Similar results were obtained by analyzing Vps21p-containing endosomes (Figure 3—figure supplement 2B). The velocity of Vps21p-containing endosomes not labeled with Vps26-mCherry (~146 ± 128 nm/s) was decreased by LatA treatment (~55 ± 42 nm/s), whereas that of ones labeled with Vps26-mCherry (~138 ± 128 nm/s) was not significantly affected (~99 ± 91 nm/s). To further confirm the association between Hse1p-labeled endosomes and the actin cytoskeleton, we labeled actin cables with tdTomato-tagged Abp140p (Yang and Pon, 2002) in wild-type cells. Simultaneous imaging revealed that Hse1p-labeled endosomes localized along, and moved on, actin cables (Figure 3E,F, and Video 1). Since two distinct sets of actin assembly-promoting machinery have been identified in yeast, the Arp2/3 complex and formins (Goode et al., 2015), we next utilized specific inhibitors toward these regulators. The Arp2/3 complex inhibitor CK-666 specifically disassembled Arp2/3 complex-dependent actin patches, whereas SMIFH2 disassembled formin-dependent actin cables (Nolen et al., 2009; Rizvi et al., 2009). As expected, SMIFH2 treatment led to actin cable disassembly, followed by a decrease in the velocity of endosomes (~60 ± 53 nm/s). In contrast, CK-666 inhibited vesicle internalization, but endosome motilities were little affected (~126 ± 123 nm/s) (Figure 3G,H, and Video 2). These results indicate that the movement of early-stage endosomes is dependent on formin-dependent actin polymerization.

Figure 3 with 2 supplements see all
Interaction of endosomes expressing Hse1-3GFP with the actin cytoskeleton.

(A and C) Movement of Hse1-3GFP-containing endosomes in living cells. Wild-type cells expressing Vps26-3mCherry and Hse1-3GFP were grown to log phase at 25°C, treated with DMSO (LatA-) (A) or 250 μM LatA (C) for 30 min at 25°C, and subsequently imaged at 1 s intervals. In the upper row the left two panels show the individual channels and the right most image shows a merged overlay of the signal from both channels in the first 19 s. The lower row shows merged images of both channels at indicated time. Yellow or red arrowheads indicate examples of Hse1p-containing endosomes that do or do not co-label with Vps26p, respectively. Scale bars, 2.5 μm. The schematic panels on the right show tracking of the endosome indicated by yellow or red arrowheads in the lower microscope images. Scale bars, 0.5 μm. (B and D) Quantification of the velocity of Hse1p-containing endosomes. Endosome velocities were acquired at 1 s intervals and categorized according to a velocity range. (E) Hse1p-containing endosomes move along actin cables. Wild-type cells expressing Hse1-3GFP and Abp140-tdTomato were grown to early to mid-logarithmic phase and each image pair was acquired simultaneously at 1 s intervals. Scale bar, 2.5 μm. (F) Higher magnification view of the boxed area in (E) at successively later time points specified in s above. Arrowheads indicate an endosome moving along an actin cable. (G) The effect of SMIFH2 or CK-666 on movement of Hse1-3GFP-containing endosomes. Wild-type cells expressing Abp140-tdTomato and Hse1-3GFP were grown to log phase at 25°C, treated with 25 μM SMIFH2 (the upper row) or 100 μM CK-666 (the lower row) for 30 min at 25°C, and subsequently imaged at 1 s intervals. The left three panels show the individual channels and their merged image for a particular time point, and the right most image shows a merged overlay of Hse1-GFP signals in 30 sec. Red arrowheads indicate examples of Hse1p-containing endosomes. Scale bars, 2.5 μm. (H) Quantification of the velocity of Hse1p-containing endosomes. Endosome velocities were acquired at 1 s intervals and categorized according to a velocity range.

https://doi.org/10.7554/eLife.10276.007
Video 1
Localization of Hse1-3GFP (left; green in merge) and Abp140-tdTomato (center; red in merge) in wild-type cells.

The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.010
Video 2
Localization of Hse1-3GFP (left; green in merge) and Abp140-Tomato (center; red in merge) in wild-type cells treated with 25 μM SMIFH2 or 100 μM CK-666.

Arrowheads indicate examples of Hse1p-labeled endosome. The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.011

Pan1p-labeled endocytic vesicles associate with actin cables in the pan1-18TA mutant

In addition to endosomes, endocytic vesicles are also known to associate with actin cables (Huckaba et al., 2004; Toshima et al., 2006), but the molecules that regulate this association remain unclear. Therefore, we examined the effect of the pan1-18TA mutation on the localization and dynamics of actin cables and endocytic vesicles. In wild-type cells, actin cables are highly dynamic polarized structures (Figure 4—figure supplement 1A and Video 3) (Yang and Pon, 2002). In contrast, the pan1-18TA mutant exhibited less polarized and more aggregated actin cable structures, some of which associated with the large actin concentrations (Figure 4—figure supplement 1A and Video 3). The movement of this Pan1p/actin aggregate in pan1-18TA was sensitive to SMIFH2 but not CK-666, suggesting that the aberrant structure associates with actin cables (Figure 4—figure supplement 1B–E). In a recent study, we showed that ~85% of endocytic vesicles were internalized along actin cables at the internalization step of endocytosis (Toshima et al., 2015). Here we show that in this step, Pan1-mCherry-labeled vesicles in wild-type cells associated with actin cables for ~4.6 s and moved on cables about 0.4 μm after internalization (Figure 4A,B,C). This association and these movements were significantly decreased by treating with 25 μM SMIFH2 (Figure 4B,C,D). Over 80% of Pan1-mCherry-labeled endocytic vesicles, even in single focal plane images, also associated with and internalized along actin cables in the pan1-18TA mutant (Figure 4E). In contrast to wild-type cells, Pan1-18TA-mCherry structures became stably associated with peripheral patches, as well as actin clumps, labeled by Abp140-3GFP (Figure 4E). Interestingly, live-cell imaging revealed that many of internalized patches labeled by Pan1-18TA-mCherry stably associated with actin cables over 30 s, and move on actin cables more than 3.0 μm (Figure 4B,C,E, and Video 4). We also wished to determine the relationship of endocytic vesicles and endosomes in the absence of Pan1 phosphorylation. In the pan1-18TA mutant, we often observed that peripheral Pan1-18TA-mCherry patches colocalized and moved together with Hse1p-labeled endosomes (Figure 4F,G, and Video 5), whereas such colocalization was rarely observed in wild-type cells (Figure 4F). These findings suggest that in the pan1-18TA mutant, endocytic vesicles and endosomes interact before fusion while they are both associated with actin cables, potentially tethering them together. Our data was collected with a wide field microscope; high-speed confocal microscopy could improve the quality of our results as it would permit 3-D analysis (Kurokawa et al., 2013). However, since some vesicles and endosomes move in a single focal plane, the simultaneous double color live cell imaging used in this study permits analysis leading to substantive biological insights.

Video 3
Localization of Abp140-3GFP in wild-type and pan1-18TA cells.

The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.012
Figure 4 with 1 supplement see all
Interaction of Pan1p-containing compartments with actin cables and endosomes.

(A) Localization of Abp140-3GFP and Pan1-mCherry in a wild-type cell. The lower panels correspond to a time series of a higher magnification view of the boxed area in the upper right image. (B) The residence time of Pan1-mCherry-labeled vesicles on an actin cable. The residence time was determined from 60 sequential two-dimensional images. = 52 vesicles for each strain. Vesicles residing on cable over 30 s are indicated as >30 in the graph. (C) Moving distance of Pan1-mCherry-labeled vesicles on actin cables. To determine each moving distance, the distance that the center of the Pan1-mCherry fluorescence moves on an actin cable was calculated based on pixel coordinates (1 pixel = 64.5 nm). = 42 vesicles for each strain. (D) Effect of the formin inhibitor SMIFH2 on the movement of Pan1-mCherry patches. Wild-type cells expressing Pan1-mCherry and Abp140-3GFP were grown to log phase at 25°C, treated with 25 μM SMIFH2 for 30 min at 25°C, and subsequently imaged at 1 s intervals. (E) The localization of Abp140-3GFP and Pan1-18TA-mCherry in a pan1-18TA cell. The lower panels are single focal plane images corresponding to a time series of a higher magnification view of the boxed area in the upper right image. Arrowheads indicate examples of Pan1p-containing compartments moving along an actin cable. Yellow or red arrowheads indicates different vesicles. Upper and middle panels show GFP and mCherry channels, respectively, and lower panel shows their merged images. (F) The localization of Hse1-3GFP and Pan1-mCherry in wild-type and pan1-18TA cells. (G) Time series of single patches in the boxed area in (F). Cells expressing Hse1-3GFP and Pan1-18TA-mCherry were grown to early to mid-logarithmic phase at 25°C in YPD medium and imaged at 1 s intervals. Scale bars, 2.5 μm.

https://doi.org/10.7554/eLife.10276.013
Video 4
Localization of Abp140-3GFP (left; green in merge) and Pan1-18TA-mCherry (center; red in merge) in pan1-18TA cells.

The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.015
Video 5
Localization of Hse1-3GFP (left; green in merge) and Pan1-18TA-mCherry (center; red in merge) in pan1-18TA cells.

Arrowheads indicate examples of colocalization. The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.016

Interaction between endocytic vesicles and actin cables upon inhibition of Prk1p

To further investigate the phosphorylation-dependent association between endocytic vesicle and actin cable, we next used an analogue-sensitive mutant of Prk1p in cells lacking Ark1p (ark1prk1-as3) (Sekiya-Kawasaki et al., 2003). This mutant shows specific sensitivity to 1NA-PP1, an ATP analogue, and enabled us to investigate the direct and immediate consequence of Prk1p inactivation for the association between endocytic vesicles and actin cables. In the mutant untreated with 1NA-PP1, Pan1-mCherry-labeled vesicles only transiently associated with actin cables, similar to wild-type cells (Figure 5A and Video 6). However, at 1 min after treatment of the mutant with 100 μM 1NA-PP1, endocytic vesicles stably associated with, and moved on actin cables. By 3 min small aggregates containing Pan1p that associated with actin cables were formed (Figure 5A and Video 6). At 10 min after 1NA-PP1 treatment, a large actin clump that stably associated with actin cables was formed in the mutant, similar to the pan1-18TA mutant (Figure 5A and Video 6; also see Figure 4C). These observations support the idea that Pan1p phosphorylation by Ark1/Prk1 kinases is necessary for the rapid dissociation of endocytic vesicles from actin cables.

Interaction between endocytic vesicles and actin cables upon inhibition of Prk1p.

(A) The left images represent single frames from movies of ark1prk1-as3 mutant cells showing merged images of the GFP (Abp140p) and the mCherry (Pan1p) channel. The ark1prk1-as3 mutant cells expressing Abp140-3GFP and Pan1-mCherry were grown to log phase at 25°C, treated with 100 μM 1NA-PP1 for the indicated time at 25°C, and subsequently imaged at 1 s intervals. A time series of single patches in the boxed area for each strain are shown in the right panels. Blue arrowheads indicate Pan1-mCherry-labeled vesicles associating with actin cables. Scale bar, 2.5 μm. (B) Localization of Abp140-3GFP and Pan1-mCherry in pan1-18TA and pan1-18TA855 cells. Scale bar, 2.5 μm. (C) Quantification of cells containing actin clumps. Cells expressing Abp140-3GFP were grown to log phase at 25°C and imaged. Data show mean ± SD from at least three experiments, with 50 cells counted for each strain per experiment.

https://doi.org/10.7554/eLife.10276.017
Video 6
Localization of Abp140-3GFP (left; green in merge) and Pan1-mCherry (center; red in merge) in ark1prk1-as3 cells untreated or treated with 100 μM 1NA-PP1.

Arrowheads indicate examples of vesicles associated with actin cables. The interval between frames is 1 s.

https://doi.org/10.7554/eLife.10276.018

Pan1p can bind directly to F-actin and its binding activity is regulated by phosphorylation through the Ark1/Prk1 kinases (Toshima et al., 2005). Thus, we next examined whether Pan1p directly mediates the interaction between vesicles and actin cables via its actin binding. To completely destroy the actin binding activity of Pan1p, we used a Pan1 C-terminal deletion mutant (pan1△855–1480), which lacks actin binding and Arp2/3-activating regions (Duncan et al., 2001; Toshima et al., 2005). Interestingly, combining the pan1-18TA and pan1△855–1480 mutations (pan1-18TA855) caused accumulation of Pan1-mCherry vesicles similar to pan1-18TA mutant, but formation of actin clumps were significantly suppressed (Figure 5B,C). The interaction between Pan1-mCherry-labeled vesicles and actin cables also decreased, but the vesicles still had an ability to bind actin cables (Figure 4D), suggesting the existence of additional actin-binding coat protein(s) that stabilize the association of vesicles with actin cables.

Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant

To be sure that this association is not merely a product of both being caught up in aberrant actin concentrations, we sought to isolate a mutant that could suppress actin clump formation in the pan1-18TA mutant. To this end, we deleted several actin-related genes involved in endocytic internalization in the pan1-18TA mutant and found three out of seven genes whose absence suppressed actin clump formation (Figure 6A,B). Deletion of Sla2p, a yeast Hip1R-related protein, in the pan1-18TA mutant decreased the fraction of cells containing actin clumps by 95% as detected by Abp1-mCherry in maximum-intensity projections of Z stacks (Figure 6—figure supplement 1A,B). These pan1-18TA sla2△ double mutants exhibited the elongated actin tails originating from non-motile endocytic sites seen in sla2△ (Kaksonen et al., 2003) (Figure 6B). Deletion of both of the yeast type I myosins, Myo3p and Myo5p, led to an 85% reduction and deletion of Sac6p, the yeast homologue of the actin filament bundling protein fimbrin, almost completely suppressed actin clump formation in the pan1-18TA mutant (Figure 6—figure supplement 1A,B). All three mutants showed a severe defect in the internalization of cortical actin patches (Kaksonen et al., 2003; 2005), resulting in the loss of actin clumps in the pan1-18TA mutant. We then wished to examine the localization of early endosomes, using Hse1-GFP as a marker, in these double and triple mutants. Interestingly, Hse1p-labeled endosomes showed a change in localization to the cell periphery in all three suppressing mutants (Figure 6B,C). We also observed that Pan1-18TA patches accumulate near Hse1p-labeled endosomes at the cell periphery in pan1-18TA sac6△ cells (Figure 6D). As reported previously (Gheorghe et al., 2008), deletion of the SAC6 gene increased the lifetime of actin patches, but around half of the endocytic vesicles were able to internalize (Figure 6—figure supplement 1C,D), indicating that the formation of endocytic vesicles is delayed but eventually occurred. In contrast, in pan1-18TA sac6△ cells, the lifetime of actin patches were significantly increased and their internalizations were markedly decreased, but ~5.3% of patches were still internalized (Figure 6—figure supplement 1D). Thus, it seems that endocytic vesicle formation is not completely blocked in pan1-18TA sac6∆ cells. To examine if endocytic vesicles are formed in this double mutant, we explored the ultrastructure of these mutants using electron microscopy. Interestingly, we observed many vesicle-like structures (~40–60 nm) accumulating around the cell periphery in pan1-18TA sac6△ cells, whereas in wild-type cells such structures were rarely detected (Figure 6—figure supplement 2A–C). We also observed that several endosome-like structures (~200–300 nm) associate with these vesicles in this mutant (Figure 6—figure supplement 2B). Quantification of these structures revealed that endosome-like structures associate with ~1–3 small vesicles in two-dimensional cross sections (Figure 6—figure supplement 2D). Similarly to pan1-18TA sac6△ cells, in pan1-18TA sla2△ cells, Hse1p-labeled endosomes were observed at the cell periphery. sla2△ cells are reported to have a severe defect in endocytic internalization, but the internalization is not completely inhibited (Raths et al., 1993) and endocytic coats are assembled on the plasma membrane (Kaksonen et al., 2003; Skruzny et al., 2012). Thus, in pan1-18TA sla2△ cells, peripheral endosomes might interact with the occasionally formed endocytic vesicles at the cell periphery or with coat proteins accumulating on the plasma membrane. Examining Hse1-GFP in overlaid time-lapse images of pan1-18TA sac6△ cells demonstrated its localization exclusively at the cell periphery and vacuolar membrane, and that peripheral endosomes are less motile (~17.8 nm/s), compared to prevacuolar ones (~189.0 nm/s) (Figure 6E,F). Since the pan1-18TA sac6△ double mutant has apparently normal late endosomes at the vacuolar membrane, similar to wild-type cells (Figure 6E), the peripheral immotile endosomes observed in the mutant are likely early endosomes. Such peripheral endosomes were rarely observed in the sac6△ mutant (Figure 6E), suggesting that they are due to the mutation of the phosphorylation sites in Pan1p and not just due to the defects in the actin cytoskeleton. LatA treatment had no effect on endosome localization in pan1-18TA sac6△ cells (Figure 6G), supporting the contention that endocytic vesicles and early endosomes stably associate, independently of actin.

Figure 6 with 2 supplements see all
Interaction between endocytic vesicles and early endosomes in the pan1-18TA mutant.

(A) Localization of Hse1-3GFP-labeled endosomes and actin structures in pan1-18TA double mutant cells. The pan1-18TA and double mutant cells expressing Hse1-3GFP and Abp1-mCherry were grown to early to mid-logarithmic phase at 25°C in YPD medium and observed by fluorescence microscopy. (B) Higher magnification view of the boxed areas in (A). Arrowheads indicate examples of Hse1p-containing endosomes at the plasma membrane. (C) Quantification of the number of Hse1p-containing endosomes localizing at the cell periphery in single focal plane images. Data show mean ± SD, with = 50 cells counted for each strain. (D) Localization of Pan1-18TA-mCherry and Hse1-3GFP in pan1-18TA sac6△ double mutant cells. (E) Movement of Hse1p-containing endosomes in pan1-18TA, sac6△, and the double mutant. Cells expressing Hse1-3GFP to visualize the endosomes were grown to log phase at 25°C, and imaged for 30 s at 1 s intervals. At time t0 = 0 s, Hse1-GFP is shown in green, at t1 = 30 s, Hse1-3GFP is shown in red. t0,t1 overlay shows overlay image of t0 and t1, and t0-t1 overlay shows overlay image of all 30 frames from t0 (0 s) to t1 (30 s). Scale bars, 2.5 μm. (F) Quantification of the velocity of Hse1p-containing endosomes at the cell periphery (PM) and the vacuolar membrane (Vac). (G) Localization of Hse1p-residing endosomes in the pan1-18TA sac6△ mutant treated with 250 μM LatA. Cells treated with 250 μM LatA for 30 min at 25°C were imaged for 30 s at 1 s intervals. At time t0 = 0 s, Hse1-GFP is shown in green, at t1 = 30 s, Hse1-3GFP is shown in red. t0,t1 overlay shows overlay image of t0 and t1. Scale bars, 2.5 μm.

https://doi.org/10.7554/eLife.10276.019

Discussion

In yeast, endocytic vesicles require actin cables to mediate their transport to the early endosome. The vesicles move in a retrograde direction, from daughter toward mother cells (Huckaba et al., 2004; Toshima et al., 2006). This is distinct from most types of transport along actin cables, such as secretion and organellar division, that move from mother to daughter cells due to the action of myosin V motor proteins (Myo2p and Myo4p). Findings that endocytic vesicle movement occurs at the same velocity and direction as that of actin cables have suggested that endocytic vesicles remain fixed on the actin cables and move as a result of actin cable flow (Girao et al., 2008; Huckaba et al., 2004).

The molecular machinery that attaches endocytic vesicles to actin cables has not yet been elucidated, although a likely candidate would be an endocytic protein that binds F-actin. Several such proteins exist (Engqvist-Goldstein and Drubin, 2003). Considering that the association between endocytic vesicles and actin cables should be transient and controllable, Pan1p is an ideal candidate to mediate this interaction. Pan1p can bind directly to F-actin with high affinity (KD<0.5 μM) and its binding activity is regulated by phosphorylation through the Ark1/Prk1 kinases (Toshima et al., 2005). In this study, we demonstrated that a Pan1-18TA △855 mutant lacking its C-terminal actin binding and Arp2/3-activating regions partially suppressed the formation of the actin clump and reduced the interaction between Pan1p-residing vesicles and actin cables. Thus, Pan1p seems to be one of the key regulators that fixes vesicles to the actin cable and then dissociate from the cable and the vesicle upon phosphorylation (Figure 7). However, the ability of vesicles to bind to actin cables was not completely lost in the mutant, implying the existence of additional actin-binding coat protein(s) that stabilize vesicle association with actin cables. Sla2p, the yeast HIP1R, and Ent1p, the yeast epsin, bind to both the plasma membrane and F-actin via their N-terminal lipid-binding domain and C-terminal actin-binding domain (Skruzny et al., 2012; Sun et al., 2005; Yang et al., 1999). A recent study showed that Sla2p and Ent1p interact redundantly with F-actin, and strains carrying a deletion of both proteins’ actin-binding domains exhibit severe a defect in endocytosis (Skruzny et al., 2012). Although the defects caused by these mutants are predominantly observed in vesicle formation, these proteins could be responsible for the residual association of endocytic vesicles with actin cables in the Pan1-18TA actin-binding mutant.

Model of the actin cable-mediated endocytic pathway.

Unphosphorylated Pan1p on an endocytic vesicle binds to actin to fix the vesicle to the actin cable. After being pinched off from the membrane, the endocytic vesicle moves into the cytosol as a result of actin cable flow, and then, interacts with the early endosome via potential tethering protein. Pan1p phosphorylation by Ark1/Prk1 kinases causes dissociation of coat proteins and the actin cable from the endocytic vesicle, making it possible for the vesicle to fuse to the endosome. This also results in the dissociation of the actin cable and the early endosome, which then moves to the vacuolar membrane, and matures into a late endosome.

https://doi.org/10.7554/eLife.10276.022

Early endosomes also associate with actin cables (Chang et al., 2003; Toshima et al., 2006), but the mechanism is still unknown. In the pan1-18TA mutant, endocytic vesicles stably associate with and move together with endosomes. This finding suggests that endocytic vesicles are capable of tethering to early endosomes, but are inefficient at fusing with them due to the inhibition of endocytic vesicle uncoating caused by Pan1p-dephosphorylation. Many tethering proteins localized at target organelles have been shown to directly interact with coat proteins of transport vesicles (Cai et al., 2007), supporting this idea. After phosphorylation of Pan1p by Ark1/Prk1 kinases, the endocytic vesicle is uncoated, making it possible to fuse to the early endosome. Thus the cycle of Pan1 phosphorylation could release endocytic vesicles from the actin cable precisely at the time of their fusion to the endosome, also allowing the endosome which is indirectly tethered to then move on and mature into a late endosome.

Our experiments may also permit a clearer ordering of the mechanistic steps of endocytosis. Many lines of evidence indicate that cargo transport from early to late endosomes is achieved by endosome maturation, which is a successive and rapid process accompanied by Rab5-Rab7 conversion (Balderhaar and Ungermann, 2013; Nordmann et al., 2010; Poteryaev et al., 2010; Rink et al., 2005). In yeast, attempts to visualize the conversion of Vps21p (yeast Rab5) to Ypt7p (yeast Rab7) on endosomes have so far been unsuccessful. Since Vps21p is localized not only at early, but also at late endosomes (Toshima et al., 2014), it is difficult to determine the point at which the early endosome ends and the late endosome begins. In the pan1-18TA mutant, localization of endosomal proteins was clearly divided into several groups, and endosomes at the early stage were more highly localized to actin clumps. This observation may be informative when considering the timing of several events comprising the endocytic pathway. For instance, subunits of the ESCRT-0, I, and II complex are highly localized but the subunit of ESCRT-III or Vps4p is localized only partially at actin clumps in the pan1-18TA mutant, suggesting that recruitment of early ESCRT components to the endosome occurs at a relatively early stage (Figure 7). Vps21p is a key regulator of early endocytic trafficking, being involved in fusion between early endosomes and the maturation of early to late endosomes (Poteryaev et al., 2010; Rink et al., 2005; Russell et al., 2012). Deletion of the VPS21 gene results in accumulation of early endosomes in the cytosol (Toshima et al., 2014). Yet Vps21p exhibited only partial localization at actin clumps, suggesting that Vps21p is recruited to the endosomal membrane after the start of ESCRT complex formation. Vps26p, a component of the retromer complex, is rarely localized at actin clumps and is predominantly localized at the vacuolar membrane, suggesting that the retromer complex mediates retrograde transport to the Golgi at the late endosome stage (Figure 7). Although Rab5-Rab7 conversion is important for endosome maturation what triggers initiation of early-to-late endosome transition is still unknown. We demonstrated that early endosomes associate with actin filaments and then change their localization to the vacuolar membrane. This suggests that dissociation of early endosomes from the actin filaments might be a trigger for the initiation of endosome maturation.

In mammalian cells, before endosomes move from the plasma membrane to the lysosome along microtubules, endosomes associate with the cortical actin cytoskeleton underlying the plasma membrane (Aschenbrenner et al., 2004; Fernandez-Borja et al., 2005). The interaction between endosomes and actin is regulated by RhoB GTPase, an upstream recruiter and activator of mammalian Dia1 and PRK1/PKN (Fernandez-Borja et al., 2005; Mellor et al., 1998). Expression of activated RhoB facilitates the association of early endosomes with cortical actin filaments, which prevents the transfer of endosomes to microtubules and inhibits further transport (Fernandez-Borja et al., 2005). The physiological importance of the interaction between endosomes and the actin cytoskeleton in mammalian cells has not been determined, but in analogy to our findings this might enable endosomes to fuse with endocytic vesicles more efficiently, at the same time preventing the progression of immature early endosomes to late endosomes.

In conclusion, our results suggest that phosphorylation of Pan1p regulates the interaction between endocytic compartments and the actin cytoskeleton. Clarifying the molecular mechanisms regulating the interaction between endocytic vesicles and endosomes, and endosomes and the actin cytoskeleton is important for elucidating the whole picture of transport from the formation of an endocytic vesicle to its fusion to an early endosome.

Materials and methods

Yeast strains, growth conditions, and plasmids

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The yeast strains used in this study are listed in the strain list (Supplementary file 1). All strains were grown in standard rich medium (YPD) or synthetic medium (SM) supplemented with 2% glucose and appropriate amino acids. C-terminal GFP or mCherry tagging of proteins was performed as described previously (Longtine et al., 1998). The pan1-18TA mutant was integrated as follows: First, to create a pan1 integration plasmid, the XmnI-DraI fragment of the PAN1 gene was cloned into pBluescript II SK, and the SalI fragment of the LEU2 gene was inserted into the SalI site 154-bp upstream of the PAN1 ORF (Toshima et al., 2005). The mutated MscI-NheI pan1-18TA fragments were used to replace the PAN1 gene in the integration plasmid. To integrate pan1 mutants at the endogenous locus, the integration plasmids were digested with SacI and XbaI, and transformed into pan1△::HIS3/PAN1 diploid strains. Integrated pan1 mutants were selected on SC plates lacking leucine and sporulated to obtain pan1-18TA mutants. Phosphorylation site mutants were constructed by a PCR-based mutagenesis protocol (Supplementary file 2).

Fluorescence microscopy

Fluorescence microscopy was performed using an Olympus IX81 microscope equipped with a x100/NA 1.40 (Olympus) objective and Orca-AG cooled CCD camera (Hamamatsu, JAPAN), using Metamorph software (Universal Imaging). Simultaneous imaging of red and green fluorescence was performed using an Olympus IX81 microscope, described above, and an image splitter (Dual-View; Optical Insights) that divided the red and green components of the images with a 565-nm dichroic mirror and passed the red component through a 630/50 nm filter and the green component through a 530/30 nm filter. These split signals were taken simultaneously with one CCD camera, described above.

Fluorescent labeling of α-factor and endocytosis assays

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Fluorescent labeling of α-factor was performed as described previously (Toshima et al., 2006). For endocytosis assays, cells were grown to an OD600 of ~0.5 in 0.5 ml YPD, briefly centrifuged, and resuspended in 20 μl SM with 5 μM Alexa Fluor-labeled α-factor. After incubation on ice for 2 hr, the cells were washed with ice-cold SM. Internalization was initiated by addition of SM containing 4% glucose and amino acids at 25°C.

Western blot assays

Preparation of cell extracts was performed as described previously (Toshima et al., 2005). In brief, the cells grown in 200 ml YPD to OD600 of 1.0 were harvested by centrifugation, washed with dH2O, resuspended to 1 ml of dH2O, drop-frozen in liquid N2, and powdered with mortar and pestle. The cell extracts were prepared using lysis buffer (50 mM Tri-HCl, pH 8.0, 150 mM NaCl, 8 M Urea, 1% Triton X-100, phosphatase inhibitor cocktail). High molecular weight proteins over 100 K molecular weight were enriched using Amicon Ultra-0.5 100 K (Millipore), and phosphorylated proteins included in the fraction were enriched using Phos-tag Agarose (NARD Institute). The enrichment of phosphorylated proteins using Phos-tag Agarose was performed as previously (Kinoshita-Kikuta et al., 2006; 2009). Immunoblot analysis was performed as described previously (Toshima et al., 2005). The chicken polyclonal antibody to GFP (GeneTex, GTX124117) was used at a dilution of 1:10000 and the HRP-conjugated rabbit polyclonal antibody to chicken IgY (Promega, G135A) at 1:10000 dilution was used as the secondary antibody. Immunoreactive proteins bands were visualized using the Western Lightning Plus ECL (PerkinElmer).

35S-labeled α-factor internalization assay

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Preparation and internalization of 35S-labeled α-factor was performed as described previously(Toshima et al., 2005). Briefly, cells were grown to an OD600 of 0.3 in 50 ml YPD, briefly centrifuged and resuspended in 4 ml YPD containing 1% (w/v) BSA, 50 mM KH2PO4, pH 6.0, and 20 μg/ml uracil, adenine, and histidine. After adding 35S-labeled α-factor, cell aliquots were withdrawn at various time points and subjected to a wash in pH 1 buffer to remove surface-bound α-factor so internal α-factor could be measured, or in pH 6 buffer to determine the total (internal and bound) α-factor. The amount of cell-associated radioactivity after each wash was determined by scintillation counting. Each experiment was performed at least three times.

Analysis of endosome motility

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Endosome motility and velocity was analyzed using the ImageJ v1.32 software package. For quantification of endosome velocity, time-lapse images were acquired at 1 s intervals. To determine the velocity, the distance traveled by each endosome in 1 s was calculated based on pixel coordinates (1 pxl = 64 nm).

Electron microscopy

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Cells sandwiched between copper disks were frozen in liquid propane at -175°C and then freeze substituted with acetone containing 2% OsO4 and 2% distilled water at -80°C for 48 hr. The samples were kept at -20°C for 4 hr and then at 4°C for 1 hr, and dehydrated in anhydrous acetone two times and 100% ethanol three times. After being infiltrated with propylene oxide (PO) two times the samples were put into a 70:30 mixture of PO and resin (Quetol-651) and then transferred to a fresh 100% resin, and polymerized at 60°C for 48 hr. The blocks were cut into 70-nm-thick sections, and the sections were mounted on copper grids. The specimens were stained with 2% uranyl acetate and Lead stain solution, and observed using a transmission electron microscope (JEM-1400Plus; JEOL).

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
    Identification of MYO4, a second class v myosin gene in yeast
    1. BK Haarer
    2. A Petzold
    3. SH Lillie
    4. SS Brown
    (1994)
    Journal of Cell Science 107:1055–1064.
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
    Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis
    1. JY Toshima
    2. C Horikomi
    3. A Okada
    4. MN Hatori
    5. M Nagano
    6. A Masuda
    7. W Yamamoto
    8. DE Siekhaus
    9. J Toshima
    (2016)
    Journal of Cell Science, 129, 10.1242/jcs.176651.
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
    Actin cable dynamics in budding yeast
    1. H-C Yang
    2. LA Pon
    (2002)
    Proceedings of the National Academy of Sciences of the United States of America 99:751–756.
    https://doi.org/10.1073/pnas.022462899
  64. 64
  65. 65
  66. 66

Decision letter

  1. Randy Schekman
    Reviewing Editor; Howard Hughes Medical Institute, University of California, Berkeley, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton" for peer review at eLife. Your submission has been favorably evaluated by Randy Schekman (Senior editor and Reviewing editor) and three reviewers.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary of the work:

The manuscript addresses a fundamental question on endocytosis: How are movements and maturation of early endocytic compartments achieved and regulated? The results presented here provide important insights into the association of earliest endosomal compartments with the actin cytoskeleton, by studying how the coat protein Pan1 impacts interactions between endocytic vesicles, early endosomes and actin.

Essential revisions:

There is much to like about this manuscript. It is well written and addresses an important problem. The data are generally of high quality. There are issues that would need to be addressed, involving some additional experimentation and quantification, before the work would be appropriate for publication in eLife. How endocytic vesicles evolve into endosomal compartments is an important unanswered question in the field. The model developed by the authors is very interesting and if true, represents a significant advance for the field.

One concern is that the main experimental technique used in this work is 2D wide-field fluorescence microscopy, which may not be the best method to analyze endosomes, which move dynamically in 3 dimensions. Some of the key data (Figure 4) in this work lack quantification. In addition, some key results using mutants (Figures 5 and 6) are a bit hard to interpret. Thus, the data are not yet adequate to support the conclusions.

Major concerns:

1) The claim that Pan1 is the key biological target for Ark1 and Prk1 kinases could use better support. The experiment done here, where 10 threonines within Sla1 are mutated to block its phosphorylation, should be backed up with some kind of experimental assessment that shows that these ten mutations completely block phosphorylation of Sla1. Without this kind of experimental evidence, it is impossible to tell whether the lack of endocytic phenotype in this mutant is due to Sla1 not being an important Ark1/Prk1 target, or if it is due to not all Ark1/Prk1 phosphorylation sites in Sla1 being mutated. Similarly, can the authors present evidence that the 18 threonines mutated in Pan1 are bona fide in vivo Ark1/Prk1 phosphorylation sites?

Nonetheless, given the similarity of the phenotype, it is likely that Ark1/Prk1 are the responsible kinases. However, that is not even relevant for the rest of their study, which deals with downstream effects of the phosphorylation. therefore, in the absence of the sort of results suggested above, the strong focus on Ark1/Prk1 in the interpretation of results as well as in Introduction and Discussion (last paragraph) should be reduced.

2) In Figure 3, the authors attempt to establish Hse1-3GFP as an early endosome marker in wild-type cells. However, Hse1 is also reported to be involved in late endosome to vacuole transport. Thus, some Hse1-3GFP puncta may not be early endosomes. In pan1-18TA mutant cells, endocytic internalization is largely defective, but it is not clear how the various endosomal compartments are affected. It does not seem reasonable to assume that Hse1-3GFP localizes only in early endosomes in this mutant. If Hse1-3GFP does not label only the early endosomes in pan1-18TA mutant, then the observation that "peripheral Pan1-18TA-mCherry patches colocalized and moved together with Hse1p-labeled endosomes," does not support the conclusion that "constitutive dephosphorylation of Pan1p, which inhibits endocytic vesicle uncoating, might lead to stable tethering between endocytic vesicles and early endosomes."

3) The last round of papers that looked at vesicle/endosome transport on actin cables was published before specific actin cytoskeleton drugs were developed, so the authors of those studies relied on LatA. Now, we have CK-666 (Arp2/3 complex inhibitor) and SMIFH2 (Formin FH2 Domain Inhibitor), so the model that vesicles move by attaching to cables and harnessing the power of actin flux could be tested more directly. Movement of these vesicles should be sensitive to SMIFH2 (or formin mutations) but not CK-666 if the model suggested in this paper is true.

4) The authors use the ESCRT0 component Hse1 as marker for early endosomes. They do not find the Rab5-orthologue Vps21, an established marker for early endosomes, to be present at significant amounts in pan1-18TA-induced actin clumps, and they further conclude form WT cells that Hse1 is present at very early endosomes, while Vps21 (and the CORVET complex) arrives later. This is in contrast to the general view that the Rab5/Vps21 marks early endosomes and is required for earliest fusion and maturation events (Zeigerer et al., Nature 2012, Cabrera et al., JBC 2013, Puchner et al., PNAS 2013 Arlt et al., MBoC 2014). The authors have previously published that initial steps in the yeast endocytic network are not dependent on Rab5, however, they have also seen that about 80% of Vps21 endosomes colocalize with Hse1 (Toshima et al., Nature Communications 2014). It is therefore surprising that Vps21 would not be able to mark the here studied earliest endosomes. Given its importance, I suggest substantiating the role of Hse1 as an early marker, provide a more precise definition of the early endosomes studied here, and discuss the results with respect to the literature on Vps21. The exclusion of Vps21 from early steps seems to be based on the quantification of actin clumps that contain the GFP-tagged protein of interest (Figure 2B). However, this does not take into account differences in the amount and lifetime of the individual proteins. I would rather suggest quantifying and comparing the percentage of GFP-fused protein that is colocalizing with actin clumps. To clarify the identity of the early endosomes that associate with actin, I suggest the measurements of endosome velocity marked by Hse1-GFP/Vps26-mCherry, with and without LatA, (subsection “Actin-dependent motility of Hse1p-residing endosomes”) to be also done with GFP-Vps21/Vps26-mCherry.

5) Data shown in Figure 4 should be quantified. One of the main claims of the paper is that the pan1-18TA mutation causes stable association of peripheral actin patches with cables. This should be quantified meticulously, and numbers for run length and amount of time spent associated with cables should be reported for patches in wild-type cells and pan1-18TA cells. Care should also be taken to define the percent of endocytic vesicles that associate with actin cables in the wild-type and mutant conditions.

6) The data shown in Figures 5 and 6 with pan1-18TA sac6∆ are hard to interpret. Deletion of SAC6 alone is known to cause substantial defects in endocytic vesicle formation and scission. Thus, it is not clear if and how endocytic vesicles are formed in pan1-18TA sac6∆ cells. For Figure 6, the authors should provide evidence that the small vesicles are endocytic vesicles and the bigger vesicles are early endosomes. In addition, a larger sample size should be analyzed for Figure 6, with quantification.

7) It seems that a more straightforward method to test the authors' model shown in Figure 7 than using pan1-18TA sac6 would be a chemical genetics approach (Bishop et al., 2001), which rapidly modulates Ark1/Prk1p kinase activity in vivo (Sekiya-Kawasaki et al.). If the authors' model is correct, upon a rapid inhibition of Ark1/Prk1 kinase activity by 1NA-PP1, Pan1p should no longer be phosphorylated and the endocytic vesicles should be fixed to the actin cables near the cell periphery. However, the data shown in Sekiya-Kawasaki et al. did not seem to suggest that this is the case.

8) The scope of the final Results section (”Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”) is a bit unclear. The authors search for mutants that suppress clump formation in pan1-18TA, to distinguish tethering of vesicles to endosomes from association of both with actin, if I understand correctly. They find three such mutants that all show peripheral, non-motile endosomes. Firstly, I miss clear evidence that these endosomes are less motile than prevacuolar ones. Second, the authors conclude from their data that these early endosomes associate with vesicles independently of actin. The problem is that sac6∆, as well as the other two suppressor deletions, are known to inhibit endocytic internalization (as the authors correctly cite) – why would there be any endocytic vesicles present? Could there be another interpretation for the presence of peripheral endosomes in these mutants? Could the vesicles seen in EM (Figure 6B) be other than primary endocytic vesicles?

9) The authors interpret their results to support the model that Pan1 mediates interaction between vesicles and actin cables (for example, Discussion, start of second paragraph). However, the experiments in Figure 4 do not distinguish between a direct role for Pan1 as the bridging molecule or an indirect role through effects on uncoating that allow for other actin-binding coat proteins to stabilize vesicle association with actin cables. One approach to address this issue might be to introduce actin-binding mutations into Pan1-18TA, such as the WH2-like domain KE mutations described in Toshima et al., 2005. Such mutations are expected to disrupt actin cable binding but not vesicle uncoating. Although these mutations suppress the actin aggregation phenotype, it should still be possible to assess vesicle interaction with actin cables. Alternatively, the actin-binding mutations could be introduced into an otherwise wild-type Pan1 and tested for effects on vesicle association with cables.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your work entitled "Pan1p regulates regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Randy Schekman as the Senior Editor and Reviewing Editor. One of the three reviewers has agreed to share his identity: Greg Payne.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript describes how primary endocytic vesicles associate with and are transported along actin cables. The transiency of this interaction appears to be regulated by phosphorylation of Pan1. Furthermore, the authors find that early endosomes also associate with actin cables. They also present evidence that tethering interactions between early endosomes and primary vesicles takes place during this phase of actin association. The authors use yeast cells expressing a Pan1-phosphorylation mutant, which retards these transient events, to dissect the actin-cable association of endosomes and vesicles. They back up their interpretations of phenotypes by experiments in wild type cells including transient drug treatments.

The authors have addressed the reviewers' major concerns in the revised manuscript. What has particularly improved the manuscript is that they clarified the role of Hse1 as an endosomal marker (and, thus, the identity of the endosomes they study), they quantified the association of vesicles and endosomes with actin, and finally, the experiments with analogue-sensitive Prk1 substantiated the phosphorylation-dependent dissociation of vesicles from cables.

We do think the manuscript is a significant contribution to our understanding of the early events in the endosomal pathway. However, several concerns remain that could be addressed by changes in the text

Essential revisions:

1) The conclusion that constitutive dephosphorylation, which inhibits uncoating, leads to stable tethering of primary vesicles to endosomes (end of subsection “Pan1p-labeled endocytic vesicles associate with actin cables in the pan1-18TA mutant”) is based on the observation that in the pan1-18TA mutant, endocytic vesicles and endosomes move together along cables. While overall, their results do suggest that endocytic vesicles and endosomes, before fusion, interact and tether while both associated to actin cables, the conclusion that dephosphorylation leads to their stable tethering, seems a bit over interpreted. In this mutant, both vesicles and endosomes are associated with actin for much longer, therefore their long-time tethering may be due to crowding or other effects, rather than the fact that dephosphorylation prevents uncoating of the vesicles.

The evidence supporting an association of endosomes with endocytic vesicles in the absence of actin is also weak. It seems clear that there is an increase in peripheral endosomes in cells lacking actin clumps (Figure 6B, C), or treated with LatA (6G), but that this localization is due to tethering with vesicles in not established. The data in 6D are not very convincing – a single static image of double mutant cells with no quantification and no comparison to single mutants. It may be a challenge for the authors to provide additional support for stable association in live cells since tracking association during movement may be difficult because of the limited motility of the peripheral endosomes. In the absence of additional data, the authors could rewrite so that association via tethering is a proposal rather than an interpretation/conclusion and either take out the tether in Figure 7 or put a question mark next to it.

Furthermore, the use of the sac6delta/pan1-18TA mutant to show that tethering of early endosomes with vesicles is not just due to actin clumping but independent of actin, is not yet entirely clear-. If I understand correctly, this finding is based on the fact that in the double mutant, early endosomes accumulate in the cell periphery, and that must be due to their association with vesicles. However, they also see endosomes in the cell periphery of sla2∆ cells, which is known to totally block endocytic vesicle formation (Kaksonen 2003, Skruzny 2012). How are these endosomes held at the cell periphery? (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”).

2) The authors failed to address the major concern that they are using 2D wide field microscopy to study endosomes, which move in three dimensions. Use of three-dimensional microscopy could improve the quality of the data presented in Figure 4 in particular. While I am disappointed that the authors still present only 2D microscopy, I believe that the data presented in Figure 4 are sufficient to support the authors' conclusions. A comment on this limitation is requested.

3) The layout of Figures 1, 4, and 6 should be changed such that the panels appear in alphabetical order. For example, panel F in Figure 6 seems to be out of place, coming after B and C but before D on the page.

4) In the text (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”), the authors claim that deletion of SLA2 leads to 95% reduction in actin clumps, but the representative image of a sla2Δ cell in Figure 6 clearly has an Abp1-labeled actin clump in the cytoplasm. In contrast, the myo3Δ/myo5Δ cell shown has no noticeable actin clump, even though the authors say in the text that clumps form more readily in this mutant. Two comments on this confusion: first, the authors should explicitly mention how they are quantifying internal clump formation; are the clumps 95% less bright in sla2Δ cells? Or do they form 95% less frequently? Second, perhaps a better representative image could be chosen, such that the numbers stated in the text are borne out in the data shown in the figure.

5) In the Discussion, first paragraph, the authors cite the vesicle formation defects in sla2 and ent1 actin-binding mutants as evidence that these proteins function before transport of endocytic vesicles on actin cables. However, predominant effects of the mutants on vesicle formation could obscure latter roles in vesicle transport and thus do not preclude such roles. Consequently, Sla2 and Ent1 could be responsible for the residual association of endocytic vesicles with actin cables in the Pan1-18TA actin-binding mutant. It might be worth editing the text in the Discussion to allow for this possibility.

https://doi.org/10.7554/eLife.10276.025

Author response

Essential revisions: There is much to like about this manuscript. It is well written and addresses an important problem. The data are generally of high quality. There are issues that would need to be addressed, involving some additional experimentation and quantification, before the work would be appropriate for publication in eLife. How endocytic vesicles evolve into endosomal compartments is an important unanswered question in the field. The model developed by the authors is very interesting and if true, represents a significant advance for the field. One concern is that the main experimental technique used in this work is 2D wide-field fluorescence microscopy, which may not be the best method to analyze endosomes, which move dynamically in 3 dimensions. Some of the key data (Figure 4) in this work lack quantification. In addition, some key results using mutants (Figures 5 and 6) are a bit hard to interpret. Thus, the data are not yet adequate to support the conclusions. 1) The claim that Pan1 is the key biological target for Ark1 and Prk1 kinases could use better support. The experiment done here, where 10 threonines within Sla1 are mutated to block its phosphorylation, should be backed up with some kind of experimental assessment that shows that these ten mutations completely block phosphorylation of Sla1. Without this kind of experimental evidence, it is impossible to tell whether the lack of endocytic phenotype in this mutant is due to Sla1 not being an important Ark1/Prk1 target, or if it is due to not all Ark1/Prk1 phosphorylation sites in Sla1 being mutated. Similarly, can the authors present evidence that the 18 threonines mutated in Pan1 are bona fide in vivo

Ark1/Prk1 phosphorylation sites?

In accordance with the reviewers’ suggestion, using Phos-tag agarose, we purified phosphorylated Sla1p from wild-type and sla1-10TA cells and examined the in vivo phosphorylation state of Sla1-10TA. In Figure 1—figure supplement 1B in the new manuscript, we show that wild-type Sla1p is phosphorylated in vivo, whereas phosphorylation of Sla1-10TA mutant is completely inhibited. We also show that Pan1p phosphorylation is also inhibited in pan1-18TA mutant (Figure 1C).

Nonetheless, given the similarity of the phenotype, it is likely that Ark1/Prk1 are the responsible kinases. However, that is not even relevant for the rest of their study, which deals with downstream effects of the phosphorylation. therefore, in the absence of the sort of results suggested above, the strong focus on Ark1/Prk1 in the interpretation of results as well as in Introduction and Discussion (last paragraph) should be reduced.

In accordance with the reviewers’ excellent suggestion, we performed experiment using analogue-sensitive mutant of Prk1p in cells lacking Ark1p (ark1prk1-as3) (please see reviewers’ comment #7). This experiment showed that transient inhibition of Prk1p’s kinase activity caused stable association of endocytic vesicles to actin cables that resembled the phenotype observed in the pan1-18TA mutant (Figure 5A). This result supports the idea that phosphorylation by Ark1/Prk1 kinases regulates interaction between vesicles and actin cables. However, since we agree the reviewers’ opinion that we emphasize Ark1/Prk1 too much without enough evidences, we modified the sentences in Introduction and Discussion as follows;

In the Introduction, the sentence “Thus, Ark1/Prk1 kinases seem to phosphorylate Pan1p to regulate the interaction between endocytic compartments and the actin cytoskeleton.” was changed to “phosphorylation of Pan1p seems to regulate the interaction between ~”.

In the Discussion, the sentence “the Ark1/Prk1 kinases phosphorylate Pan1p to regulate the interaction ~” was changed to “phosphorylation of Pan1p regulates ~”.

2) In Figure 3, the authors attempt to establish Hse1-3GFP as an early endosome marker in wild-type cells. However, Hse1 is also reported to be involved in late endosome to vacuole transport. Thus, some Hse1-3GFP puncta may not be early endosomes. In pan1-18TA mutant cells, endocytic internalization is largely defective, but it is not clear how the various endosomal compartments are affected. It does not seem reasonable to assume that Hse1-3GFP localizes only in early endosomes in this mutant. If Hse1-3GFP does not label only the early endosomes in pan1-18TA mutant, then the observation that "peripheral Pan1-18TA-mCherry patches colocalized and moved together with Hse1p-labeled endosomes," does not support the conclusion that "constitutive dephosphorylation of Pan1p, which inhibits endocytic vesicle uncoating, might lead to stable tethering between endocytic vesicles and early endosomes."

We agree with the reviewer that some Hse1-3GFP puncta may not be early endosomes. As we showed in Figure 3—figure supplement 1C, D, Hse1p is widely localized from early to late endosomes and partially colocalized with Vps26p at late endosomes. We also showed that similar levels of Hse1p colocalized with Vps26p in pan1-18TA mutant (Figure 3—figure supplement 1D, H). Thus, we changed the sentence “constitutive dephosphorylation of Pan1p, which inhibits endocytic vesicle uncoating, might lead to stable tethering between endocytic vesicles and early endosomes.” to “~ stable tethering between endocytic vesicles and endosomes.”

3) The last round of papers that looked at vesicle/endosome transport on actin cables was published before specific actin cytoskeleton drugs were developed, so the authors of those studies relied on LatA. Now, we have CK-666 (Arp2/3 complex inhibitor) and SMIFH2 (Formin FH2 Domain Inhibitor), so the model that vesicles move by attaching to cables and harnessing the power of actin flux could be tested more directly. Movement of these vesicles should be sensitive to SMIFH2 (or formin mutations) but not CK-666 if the model suggested in this paper is true.

We examined the effects of CK-666 and SMIFH2 on the movement of endocytic vesicles and endosomes. As shown in Figure 3G, H and 4B, D, E, we found that the movement of endocytic vesicles and endosomes are sensitive to SMIFH2 but not CK-666, as the reviewer suggested.

4) The authors use the ESCRT0 component Hse1 as marker for early endosomes. They do not find the Rab5-orthologue Vps21, an established marker for early endosomes, to be present at significant amounts in pan1-18TA-induced actin clumps, and they further conclude form WT cells that Hse1 is present at very early endosomes, while Vps21 (and the CORVET complex) arrives later. This is in contrast to the general view that the Rab5/Vps21 marks early endosomes and is required for earliest fusion and maturation events (Zeigerer et al., Nature 2012, Cabrera et al., JBC 2013, Puchner et al., PNAS 2013 Arlt et al., MBoC 2014). The authors have previously published that initial steps in the yeast endocytic network are not dependent on Rab5, however, they have also seen that about 80% of Vps21 endosomes colocalize with Hse1 (Toshima et al., Nature Communications 2014). It is therefore surprising that Vps21 would not be able to mark the here studied earliest endosomes. Given its importance, I suggest substantiating the role of Hse1 as an early marker, provide a more precise definition of the early endosomes studied here, and discuss the results with respect to the literature on Vps21. The exclusion of Vps21 from early steps seems to be based on the quantification of actin clumps that contain the GFP-tagged protein of interest (Figure 2B). However, this does not take into account differences in the amount and lifetime of the individual proteins. I would rather suggest quantifying and comparing the percentage of GFP-fused protein that is colocalizing with actin clumps. To clarify the identity of the early endosomes that associate with actin, I suggest the measurements of endosome velocity marked by Hse1-GFP/Vps26-mCherry, with and without LatA, (subsection “Actin-dependent motility of Hse1p-residing endosomes”) to be also done with GFP-Vps21/Vps26-mCherry.

To determine the relative localization of Hse1p and Vps21p at early stages of endocytosis, we slowed endocytic traffic by removing glucose from the culture medium (Aoh et al., MBoC, 2011), and compared their localization with internalized A594-α-factor. Interestingly, we found that Hse1p colocalizes with A594-α-factor at slightly higher amounts than Vps21p at 10 min after α-factor internalization (Figure 3—figure supplement 2A). This result is consistent with a recent study by Arlt et al. (MBoC, 2015). In the paper, they carefully compared the recruitment timing of several endosomal proteins, including ESCRT subunits and Vps21p and reported that an ESCRT-I subunit, Vps23, is recruited to endosomes earlier than Vps21p. We also quantified the percentage of GFP-fused Vps21p that colocalizes with actin clumps, as the reviewer suggested, and found only ~8% of GFP-Vps21p is localized to actin clumps whereas ~22% of Hse1-GFP is localized to actin clumps (data not shown in the manuscript). Thus, it seems to be reasonable that Hse1p, another ESRCT-I subunit, is recruited to early endosomes before Vps21p, and that Hse1p localizes to actin clumps more than Vps21p.

Additionally, in accordance with the reviewers’ suggestion, we examined the velocity of endosomes marked by GFP-Vps21 with or without Vps26-mCherry. Similar to Hse1p-residing endosomes, the velocity of GFP-Vps21 endosomes not labeled with Vps26-mCherry was decreased by LatA treatment, whereas those labeled with Vps26-mCherry was not significantly affected (Figure 3—figure supplement 2B). Taken with the previous results (colocalization with Vps21p and recruitment of ESCRT-1 complex to early endosomes), we concluded that Hse1p is suitable as an early endosome marker, as well as Vps21p.

5) Data shown in Figure 4 should be quantified. One of the main claims of the paper is that the pan1-18TA mutation causes stable association of peripheral actin patches with cables. This should be quantified meticulously, and numbers for run length and amount of time spent associated with cables should be reported for patches in wild-type cells and pan1-18TA cells. Care should also be taken to define the percent of endocytic vesicles that associate with actin cables in the wild-type and mutant conditions.

According to the reviewers’ suggestion, we added quantified data in Figure 4D and 4E. With regard to percent of endocytic vesicles that associate with actin cables, we recently reported that over 80% of actin patches were internalized actin cables at the internalization step of endocytosis (Toshima et al., JCS, 2015). We also examined their association in pan1-18TA mutant, and found that over 80% of Pan1-mCherry-labeled endocytic vesicles also associated with and internalized along actin cables in pan1-18TA mutant. To explain this result, we have added some sentences to the text (subsection “Pan1p-labeled endocytic vesicles associate with actin cables in the pan1-18TA mutant”).

6) The data shown in Figures 5 and 6 with pan1-18TA sac6∆ are hard to interpret. Deletion of SAC6 alone is known to cause substantial defects in endocytic vesicle formation and scission. Thus, it is not clear if and how endocytic vesicles are formed in pan1-18TA sac6∆ cells. For Figure 6, the authors should provide evidence that the small vesicles are endocytic vesicles and the bigger vesicles are early endosomes. In addition, a larger sample size should be analyzed for Figure 6, with quantification.

To examine the defects of endocytic vesicle formation and scission quantitatively in sac6△ and pan1-18TA sac6∆ mutant, we quantified actin patch lifetime and percentage of vesicle internalization. As reported previously (Gheorghe et al., 2008), deletion of the SAC6 gene increased actin patch lifetime, but around half of endocytic vesicles were able to move inside the cells, indicating that though the formation of endocytic vesicles is delayed it eventually occurred (Figure 6—figure supplement 1C, D). In contrast, in pan1-18TA sac6△ cells, the lifetime of actin patches were significantly increased and their internalizations were rarely observed. Yet ~5.3% of patches were still internalized (Figure 6—figure supplement 1C, D) supporting the conclusion that endocytic vesicle formation is not completely blocked in pan1-18TA sac6∆ cells.

We also performed immuno-EM to identify the small vesicles and the bigger vesicles observed in the EM images as endocytic vesicles and early endosomes, but we could not obtain a clear result because our antibodies did not work on the EM samples, probably due to the low expression of Pan1p or Hse1p on the vesicles. Thus, as reviewer #2 suggested in comment #5, we moved the Figure to the supplementary file (Figure 6—figure supplement 2) and de-emphasized the result in the new manuscript (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”). In addition, we have added quantified data in Figure 6—figure supplement 2C and D.

7) It seems that a more straightforward method to test the authors' model shown in Figure 7 than using pan1-18TA sac6 would be a chemical genetics approach (Bishop et al., 2001), which rapidly modulates Ark1/Prk1p kinase activity in vivo (Sekiya-Kawasaki et al.). If the authors' model is correct, upon a rapid inhibition of Ark1/Prk1 kinase activity by 1NA-PP1, Pan1p should no longer be phosphorylated and the endocytic vesicles should be fixed to the actin cables near the cell periphery. However, the data shown in Sekiya-Kawasaki et al.

did not seem to suggest that this is the case.

In accordance with the reviewers’ suggestion, we performed an experiment using an analogue-sensitive mutant of Prk1p in cells lacking Ark1p (ark1prk1-as3). This mutant enabled us to investigate the direct and immediate consequence of Prk1p inactivation for the interaction between endocytic vesicles and actin cables. As shown in Figure 5A, we found that, at 1 min after treatment of the mutant with 100 μM 1NA-PP1, endocytic vesicles stably associated with, and moved on actin cables. Then, small aggregates containing Pan1p that associated with actin cables were formed by 3 min, and a large actin clump that stably associates with actin cables was formed in the mutant after 10 min (Figure 5A). These observations support the idea that Pan1p phosphorylation by Ark1/Prk1 kinases promotes dissociation of endocytic vesicles from actin cables.

8) The scope of the final Results section (”Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”) is a bit unclear. The authors search for mutants that suppress clump formation in pan1-18TA, to distinguish tethering of vesicles to endosomes from association of both with actin, if I understand correctly. They find three such mutants that all show peripheral, non-motile endosomes. Firstly, I miss clear evidence that these endosomes are less motile than prevacuolar ones.

We have added quantified data for the endosome velocity in Figure 6F.

Second, the authors conclude from their data that these early endosomes associate with vesicles independently of actin. The problem is that sac6∆, as well as the other two suppressor deletions, are known to inhibit endocytic internalization (as the authors correctly cite) – why would there be any endocytic vesicles present? Could there be another interpretation for the presence of peripheral endosomes in these mutants? Could the vesicles seen in EM (Figure 6B) be other than primary endocytic vesicles?

To examine of if endocytic vesicles are formed in pan1-18TA sac6∆ cells, we examined the lifetime and dynamics of actin patches in sac6∆ and pan1-18TA sac6∆ cells (please see the response to comment #6). As shown in Figure 6—figure supplement 1C, D, the lifetime of actin patches were significantly increased and their internalizations were rarely observed, but ~5.3% of patches were still internalized. Thus, it seems that endocytic vesicle formation is not completely blocked in pan1-18TA sac6∆ cells. With regard to the EM data, we have moved the Figure to the supplementary file and de-emphasized the result, because we could not confirm the exact identity of the vesicle-like and endosome-like structures through antibody stains.

9) The authors interpret their results to support the model that Pan1 mediates interaction between vesicles and actin cables (for example, Discussion, start of second paragraph). However, the experiments in Figure 4 do not distinguish between a direct role for Pan1 as the bridging molecule or an indirect role through effects on uncoating that allow for other actin-binding coat proteins to stabilize vesicle association with actin cables. One approach to address this issue might be to introduce actin-binding mutations into Pan1-18TA, such as the WH2-like domain KE mutations described in Toshima et al., 2005. Such mutations are expected to disrupt actin cable binding but not vesicle uncoating. Although these mutations suppress the actin aggregation phenotype, it should still be possible to assess vesicle interaction with actin cables. Alternatively, the actin-binding mutations could be introduced into an otherwise wild-type Pan1 and tested for effects on vesicle association with cables.

To determine whether Pan1p directly mediates the interaction between endocytic vesicles and actin cables, we prepared the Pan1-18TA mutant lacking its C terminal actin binding and Arp2/3-activating regions (Duncan et al., NCB, 2001; Toshima et al., NCB, 2005). Interestingly, as the reviewer expected, pan1-18TA855 caused accumulation of vesicles containing Pan1p similar to that seen in the pan1-18TA mutant, but the formation of actin clumps was significantly suppressed (Figure 5B and 5C). The interaction between vesicles and actin cables also decreased, but the ability of endocytic vesicles to bind to actin cables was not completely lost (Figure 4D). Thus, it seems that other actin-binding coat protein(s) that stabilizes vesicle association with actin cables exist, in addition to Pan1p.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Essential revisions: 1) The conclusion that constitutive dephosphorylation, which inhibits uncoating, leads to stable tethering of primary vesicles to endosomes (end of subsection “Pan1p-labeled endocytic vesicles associate with actin cables in the pan1-18TA mutant”) is based on the observation that in the pan1-18TA mutant, endocytic vesicles and endosomes move together along cables. While overall, their results do suggest that endocytic vesicles and endosomes, before fusion, interact and tether while both associated to actin cables, the conclusion that dephosphorylation leads to their stable tethering, seems a bit over interpreted. In this mutant, both vesicles and endosomes are associated with actin for much longer, therefore their long-time tethering may be due to crowding or other effects, rather than the fact that dephosphorylation prevents uncoating of the vesicles. The evidence supporting an association of endosomes with endocytic vesicles in the absence of actin is also weak. It seems clear that there is an increase in peripheral endosomes in cells lacking actin clumps (Figure 6B, C), or treated with LatA (6G), but that this localization is due to tethering with vesicles in not established. The data in 6D are not very convincing

a single static image of double mutant cells with no quantification and no comparison to single mutants. It may be a challenge for the authors to provide additional support for stable association in live cells since tracking association during movement may be difficult because of the limited motility of the peripheral endosomes. In the absence of additional data, the authors could rewrite so that association via tethering is a proposal rather than an interpretation/conclusion and either take out the tether in Figure 7 or put a question mark next to it.

In accordance with the reviewers’ suggestion, we rewrote the sentence which said “constitutive dephosphorylation, which inhibits uncoating, leads to stable tethering of primary vesicles to endosomes.” to “in the pan1-18TA mutant, endocytic vesicles and endosomes interact before fusion while they are both associated with actin cables, potentially tethering them together.” We also modified Figure 7 as reviewer suggested, indicating a protein as a “potential tethering protein” in the legend and placing a question mark next to it in the figure.

Furthermore, the use of the sac6∆ /pan1-18TA mutant to show that tethering of early endosomes with vesicles is not just due to actin clumping but independent of actin, is not yet entirely clear-. If I understand correctly, this finding is based on the fact that in the double mutant, early endosomes accumulate in the cell periphery, and that must be due to their association with vesicles. However, they also see endosomes in the cell periphery of sla2∆ cells, which is known to totally block endocytic vesicle formation (Kaksonen 2003, Skruzny 2012). How are these endosomes held at the cell periphery? (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”).

As the reviewers’ suggested, sla2△ cells are reported to have a severe defect in endocytic internalization, but the internalization is not completely blocked at 24oC as shown in Figure 1 (Raths et al., JCB, 1993). Thus, similarly to pan1-18TA sac6△ cells, endocytic vesicle formation might not be completely blocked in pan1-18TA sla2∆ cells and peripheral endosomes could interact with those endocytic vesicles. As another possibility, in sla2△ cells, although endocytic internalization is severely inhibited, endocytic coats are assembled on the plasma membrane (Kaksonen et al., Cell, 2003; Skruzny et al., PNAS, 2012). Thus, peripheral endosomes might interact with coat proteins accumulating on the plasma membrane in pan1-18TA sla2△ cells. To explain about this, we have added some sentences in the Results section (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”).

2) The authors failed to address the major concern that they are using 2D wide field microscopy to study endosomes, which move in three dimensions. Use of three-dimensional microscopy could improve the quality of the data presented in Figure 4 in particular. While I am disappointed that the authors still present only 2D microscopy, I believe that the data presented in Figure 4 are sufficient to support the authors' conclusions. A comment on this limitation is requested.

In accordance with the reviewers’ suggestion, we have added sentences in the Results section (subsection “Pan1p-labeled endocytic vesicles associate with actin cables in the pan1-18TA mutant”).

3) The layout of Figures 1, 4, and 6 should be changed such that the panels appear in alphabetical order. For example, panel F in Figure 6 seems to be out of place, coming after B and C but before D on the page.

We modified those Figures to arrange all panels in alphabetical order.

4) In the text (subsection “Interaction between endocytic vesicles and early endosomes in the pan1-18TA phosphorylation site mutant”), the authors claim that deletion of SLA2 leads to 95% reduction in actin clumps, but the representative image of a sla2Δ cell in Figure 6 clearly has an Abp1-labeled actin clump in the cytoplasm. In contrast, the myo3Δ/myo5Δ cell shown has no noticeable actin clump, even though the authors say in the text that clumps form more readily in this mutant. Two comments on this confusion: first, the authors should explicitly mention how they are quantifying internal clump formation; are the clumps 95% less bright in sla2Δ

cells? Or do they form 95% less frequently? Second, perhaps a better representative image could be chosen, such that the numbers stated in the text are borne out in the data shown in the figure.

To mention how we quantified internal clump formation described in Figure 6—figure supplement 1A and 1B, we have added a sentence in the legend of the Figure. We also changed the sentence “Deletion of Sla2p, a yeast Hip1R-related protein, in the pan1-18TA mutant decreased the formation of actin clumps by 95%~” to “Deletion of Sla2p, a yeast Hip1R-related protein, in the pan1-18TA mutant decreased the fraction of cells containing actin clumps by 95% ~”. We have also replaced the micrographs in Figure 6A (sla2△ and myo3/5△ cells) and Figure 6—figure supplement 1A (myo3/5△ cells) to better ones.

5) In the Discussion, first paragraph, the authors cite the vesicle formation defects in sla2 and ent1 actin-binding mutants as evidence that these proteins function before transport of endocytic vesicles on actin cables. However, predominant effects of the mutants on vesicle formation could obscure latter roles in vesicle transport and thus do not preclude such roles. Consequently, Sla2 and Ent1 could be responsible for the residual association of endocytic vesicles with actin cables in the Pan1-18TA actin-binding mutant. It might be worth editing the text in the Discussion to allow for this possibility.

According to the reviewers’ suggestion, we have changed the sentence from “~ these proteins seem to function at the steps before the transport of endocytic vesicles along the actin cable.” to “~ these proteins could be responsible for the residual association of endocytic vesicles with actin cables in the Pan1-18TA actin-binding mutant.”

https://doi.org/10.7554/eLife.10276.026

Article and author information

Author details

  1. Junko Y Toshima

    1. Department of Liberal Arts, Tokyo University of Technology, Tokyo, Japan
    2. Research Center for RNA Science, Tokyo University of Science, Tokyo, Japan
    Contribution
    JYT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Eri Furuya

    Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    EF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  3. Makoto Nagano

    1. Research Center for RNA Science, Tokyo University of Science, Tokyo, Japan
    2. Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    MN, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  4. Chisa Kanno

    Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    CK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  5. Yuta Sakamoto

    Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    YS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  6. Masashi Ebihara

    Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    ME, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  7. Daria Elisabeth Siekhaus

    Institute of Science and Technology Austria, Klosterneuburg, Austria
    Contribution
    DES, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  8. Jiro Toshima

    1. Research Center for RNA Science, Tokyo University of Science, Tokyo, Japan
    2. Department of Biological Science and Technology, Tokyo University of Science, Tokyo, Japan
    Contribution
    JT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    jtosiscb@rs.noda.tus.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3264-9843

Funding

Japan Society for the Promotion of Science (KAKENHI GRANT #26440067)

  • Junko Y Toshima

Japan Society for the Promotion of Science London (KAKENHI GRANT #25440054)

  • Jiro Toshima

European Union Grant (PCIG12-GA-2012-334077)

  • Daria Elisabeth Siekhaus

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank A Masuda and N Yoshida for construction of plasmids and strains, and C Horikomi for analyzing data. This work was supported by JSPS KAKENHI GRANT #26440067 and a Takeda Science Foundation grant to JYT, as well as JSPS KAKENHI GRANT #25440054 and a Takeda Science Foundation grant to JT. Daria E Siekhaus is supported by EU grant PCIG12-GA-2012-334077.

Reviewing Editor

  1. Randy Schekman, Howard Hughes Medical Institute, University of California, Berkeley, United States

Publication history

  1. Received: July 22, 2015
  2. Accepted: February 1, 2016
  3. Version of Record published: February 25, 2016 (version 1)

Copyright

© 2016, Toshima et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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