Correspondence to Kendall J. Blumer: kblumer{at}cellbio.wustl.edu
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Introduction |
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Internalized receptors use several mechanisms to move within minutes from the plasma membrane to lysosomes. Motor proteins such as myosin VI and myosin V can move endosomes along actin filaments near the cell cortex (Schott et al., 1999; Aschenbrenner et al., 2003; Hasson, 2003; Soldati, 2003), and kinesin and dynein can move endosomes along microtubules over longer distances (Aniento et al., 1993; Apodaca, 2001; Bananis et al., 2003). Actin polymerization can also power endosome movement (Taunton et al., 2000), similar to mechanisms that transport intracellular pathogens (Loisel et al., 1999), macropinosomes (Merrifield et al., 2001; Seastone et al., 2001), and the insulin-responsive glucose transporter Glut4 (Kanzaki et al., 2001). These actin polymerization-dependent transport mechanisms are thought to use the Arp2/3 complex to nucleate ongoing and continuous assembly of branched actin filament networks on organelle membranes (reviewed in Schafer, 2002). The WASp/SCAR/WAVE family of proteins are potent activators of the Arp2/3 complex (Pollard and Borisy, 2003), and have been implicated in promoting motility of endosomes and other organelles (Taunton et al., 2000; Southwick et al., 2003). Indeed, we have shown that Las17, the sole WASp homologue of yeast, is required for motility of endosomes containing the GPCR Ste2 (Chang et al., 2003).
Actin dynamics may power organelle motility by other mechanisms as suggested by studies of endocytosis in yeast. In one model, actin polymerization-driven internalization imparts momentum that carries endosomes through the cytoplasm (Kaksonen et al., 2003). In support of this model, the WASp homologue Las17 and the actin binding protein Abp1 are detected on cortical actin patches as they internalize but are depleted from endosomes moving away from the cortex (Kaksonen et al., 2003). However, the distance or speed with which endosomes could travel through the viscous cytoplasm by such a mechanism is unclear. In a second mechanism, a subpopulation of endosomes have been shown to bind cytoplasmic F-actin cables that treadmill, transporting endosomes to the lysosome-like vacuole (Huckaba et al., 2004).
Mechanisms regulating actin polymerization-dependent endosome and organelle motility have emerged from studies of WASp/SCAR/WAVE proteins. WASp proteins are autoinhibited and form complexes with accessory proteins including WIP and TOCA-1 (Moreau et al., 2000; Ho et al., 2004). Phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PI4,5P2; PIP2) in concert with GTP-loaded Cdc42 or Rac1 can bind and activate WASp in vitro (Kanzaki et al., 2001; Benesch et al., 2002; Sokac et al., 2003) by exposing the WASp COOH-terminal Arp2/3 complex activating region (WCA) domain (reviewed in Higgs and Pollard, 2001). Furthermore, overexpression of phosphatidylinositol 4-phosphate (PI4P)5-kinase induces N-WASPdependent motility of endosomes and other organelles (Rozelle et al., 2000; Taunton et al., 2000). Regulation of SCAR/WAVE proteins is less well understood. SCAR/WAVE proteins are constitutively active, but can be stabilized in inactive complexes with accessory proteins (Nap1, Abi2, HSPC300; Eden et al., 2002; Innocenti et al., 2004; Steffen et al., 2004).
Here we have addressed whether endosomes in yeast move by the momentum gained upon actin polymerization-driven abscission from the plasma membrane or by continuously polymerizing actin on endosome membranes after abscission. We also address how the yeast WASp homologue Las17 is regulated to drive endosome motility. Our findings indicate that endosome motility requires continuous actin polymerization on endosomes, and that Lsb6, a type II phosphatidylinositol 4-kinase (PI 4-kinase) of previously unknown function, regulates endosome motility by interacting with Las17 rather than by catalyzing PI4P synthesis.
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Results |
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To determine whether expression of Ste2-WCA or Hxt1-WCA could rescue the endosome motility defect of las17WCA mutants, we introduced Ste2-GFP into these cells as an endosome marker (Stefan and Blumer, 1999) and analyzed endosome movement in these transformants by making single focal plane time-lapse movies. Endosome (n = 2550) motility in each mutant was measured by calculating average speed, distribution of speeds, and by tracing paths of endosome motion over a 510-s period. Using these methods we showed previously that endosomes labeled with Ste2-GFP in wild-type cells move with an average speed of
0.2 µm/s, travel along paths of various length with marked changes in direction, and often leave the plane of focus within a few seconds (Chang et al., 2003).
Ste2-WCA and Hxt1-WCA fusions had strikingly different effects on the motility of endosomes labeled with Ste2-GFP in a las17WCA mutant (Table I). Because Ste2 efficiently and constitutively forms homooligomers (Overton and Blumer, 2000), Ste2-WCA and Ste2-GFP will traffic together during endocytosis. Using this approach, we found that expression of Ste2-WCA fully rescued the endosome motility defect of a las17
WCA mutant. Rescued cells displayed an average endosome speed of 0.18 ± 0.01 µm/s relative to that of 0.08 ± 0.01 µm/s in empty vector control cells. As occurs in wild-type cells, endosomes in rescued cells traveled along paths that were long and changed direction, often leaving the plane of focus after a few seconds. In contrast, although Hxt1-WCA was highly overexpressed on the plasma membrane in a pattern indistinguishable from Ste2 (Fig. S1 B available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1), it failed to rescue the endosome motility defect of las17
WCA mutants (Table I). These results are illustrated in Video S1 (endosome motility in a las17
WCA cell + vector), Video S2 (endosome motility in a las17
WCA cell + Ste2-WCA), and Video S3 (endosome motility in a las17
WCA cell + Hxt1-WCA; videos available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1).
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The type II PI 4-kinase Lsb6 is required for endosome motility
Little is known about the mechanisms that regulate Las17. Purified Las17 appears to be constitutively active, yet it can be inhibited in vitro by the SH3-domain proteins Bbc1 and Sla1 (Rodal et al., 2003). It remains unknown whether Las17 in vivo is constitutively active or regulated by stimulatory or inhibitory proteins or ligands.
To identify proteins that may activate or recruit Las17 and thereby promote motility of Ste2-containing endosomes, we analyzed mutants lacking proteins that were identified previously in a two-hybrid screen as Las17-binding proteins (Lsb; Madania et al., 1999). These proteins include Lsb1 and 2 (SH3 domaincontaining proteins similar to Grb2 or Grap2), Lsb3 and Lsb4 (SH3 domaincontaining proteins similar to intersectin 1), Lsb5 (VRS domaincontaining protein like HRS), and Lsb6 (the single type II PI 4-kinase homologue in yeast). Endosome motility was assayed as described above in null mutants lacking each of these Las17-interacting proteins, and in double mutants lacking pairs of closely related proteins (Lsb1 and Lsb2, or Lsb3 and Lsb4).
Of the six single mutants and two double mutants analyzed, lsb6 mutants displayed significant impairment of endosome motility relative to wild-type cells. Endosomes in lsb6
and wild-type cells moved with average speeds of 0.09 ± 0.01 µm/s and 0.19 ± 0.02 µm/s, respectively (Table I). The average speed of endosome motility in lsb6
mutants was similar to that observed in cells expressing Las17 lacking its COOH-terminal WCA domain (las17
WCA, 0.09 ± 0.01 µm/s) or in wild-type cells treated with the actin-depolymerizing drug latrunculin A (0.08 ± 0.04 µm/s; Chang et al., 2003). In lsb6
mutants, the distribution of endosome speeds was shifted to lower values (Fig. 2 C), and endosomes moved along abnormally short paths that remained within the plane of focus for many seconds (Fig. 2 B). Impaired endosome motility in lsb6
mutants was rescued by expression of the wild-type LSB6 gene on a single-copy plasmid (compare Video S6 [endosome motility in an lsb6
cell] with Video S7 [endosome motility in an lsb6
cell + pLSB6]; videos available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1). These results were confirmed by performing automated particle tracking and analyzing the data in MSD plots (Fig. S2 A). These results indicated that Lsb6 is required for motility of Ste2-containing endosomes. This is the first phenotype caused by the absence of the sole type II PI 4-kinase of yeast.
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PI 4-kinase activity of Lsb6 is dispensable for endosome motility
Lsb6 is the only type II PI 4-kinase in yeast (Han et al., 2002; Shelton et al., 2003). However, yeast also possesses two type III PI 4-kinases, Stt4 and Pik1, each of which is essential for cell growth (Audhya et al., 2000). Whereas Lsb6 synthesizes a small fraction (5%) of the total PI4P pool, the remainder of the PI4P pool is produced by Stt4 on the plasma membrane and by Pik1 on Golgi membranes (Han et al., 2002). These plasma membrane and Golgi PI4P pools have distinct functions because overexpression of Stt4 does not rescue the phenotype of pik1 mutants and vice versa (Han et al., 2002).
To determine whether Lsb6 synthesizes PI4P to promote endosome motility, we generated and analyzed several point mutants defective in PI 4-kinase activity. Like other type II PI 4-kinases, the kinase domain of Lsb6 is interrupted by a linker such that motifs 1, 2, 3, and 4 of the catalytic domain are located in the NH2-terminal portion of the molecule and motifs 6 and 7 in the COOH-terminal region (Fig. 3 A and Fig. S3). Flanking the two kinase subdomains are noncatalytic NH2- and COOH-terminal extensions. Studies of the rat type II PI 4-kinase have identified residues required specifically for catalysis; substitution of any of these residues eliminates enzyme activity (Barylko et al., 2002). Accordingly, we targeted the equivalent amino acids either singly or together in NH2-terminally HA-tagged Lsb6 to yield the following mutants: K192M, D387A, N392A, D413A, and a quadruple mutant (4KD) bearing all four of these substitutions (Fig. 3 A). Each mutant form of HA-Lsb6 of the expected molecular mass was well expressed (Fig. 3 B). We used assay conditions optimized to detect the PI 4-kinase activity of Lsb6 and to minimize activity of the other PI 4-kinases Stt4 and Pik1 (Han et al., 2002). Under these conditions, we found that extracts from cells expressing wild-type HA-Lsb6 showed robust PI 4-kinase activity relative to the low level of activity detected in extracts from lsb6 cells expressing vector alone (Fig. 3 C). In contrast, extracts derived from cells expressing any of the kinase domain point mutants lacked PI 4-kinase activity above the background (Fig. 3 C).
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PI4P generated by Stt4 or Pik1 is dispensable for endosome motility
Although the PI 4-kinase activity of Lsb6 is dispensable for endosome motility, the preceding experiments could not exclude a role for PI4P production by Stt4 or Pik1. Accordingly, we examined endosome motility in a pik1 or stt4 temperature-sensitive mutant or in an lsb6 stt4-ts double mutant. The pik1 and stt4 temperature-sensitive mutant phenotypes were confirmed according to published assays that scored aberrant vacuolar morphology or actin patch depolarization, respectively (Audhya et al., 2000). Inactivation of these PI 4-kinases by temperature shift, however, did not result in defective endosome motility (unpublished data). These results suggested that Pik1 and Stt4 are dispensable for endosome motility.
As a further means of exploring the role of phosphoinositide synthesis in endosome motility, we examined temperature-sensitive mss4 mutants, which are defective in the sole PI4P 5-kinase in yeast. At nonpermissive temperature, the mss4 mutant lost PI4P 5-kinase activity, as indicated by cytosolic localization of a PI4,5P2 sensor (PLC-PH-GFP; Wild et al., 2004). Under these conditions, defects in endosome motility were not observed (unpublished data). Therefore, PI4,5P2 produced by Mss4 is dispensable for endosome motility.
The NH2-terminal half of Lsb6 mediates endosome motility and Las17 interaction
Because the kinase activities of Lsb6 and other PI 4-kinases are dispensable for endosome motility, we hypothesized that Lsb6 promotes endosome motility by interacting with Las17. To test this hypothesis, we determined which domains of Lsb6 are necessary and sufficient for endosome motility and Las17 interaction.
Accordingly, we generated a series of deletion mutants of HA-Lsb6 expressed from plasmids in lsb6 cells (Fig. 4). All Lsb6 deletion constructs exhibited undetectable PI 4-kinase activity (unpublished data). Analysis of endosome motility in lsb6
mutants expressing these constructs indicated that the NH2-terminal region flanking the catalytic domain was necessary for endosome motility (Table I). This result is illustrated by comparing Video S9 (endosome motility in an lsb6
cell + pHA-Lsb6
N-terminus) and Video S7 (endosome motility in an lsb6
cell + pLsb6). In contrast, deletion of other regions of Lsb6 did not affect endosome motility (Table I). This result is illustrated by comparing Video S10 (endosome motility in an lsb6
cell + pHA-Lsb6
C-terminus) and Video S7 (endosome motility in an lsb6
cell + pLsb6). Both kinds of results were confirmed quantitatively by using automated particle tracking and MSD plots (Fig. S2). Expression of the NH2-terminal domain of Lsb6 rescued endsome motility less well than a construct containing both the NH2-terminal domain and the first half of the kinase domain (Table I). Taken together, these results indicated that the region of Lsb6 containing the NH2-terminal domain and first half of the kinase domain is necessary and sufficient for full activity.
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Discussion |
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Actin polymerization-driven endosome motility
Extensive evidence has demonstrated that actin assembly in yeast is critical for endocytic internalization and transport, and that endosomes do not use myosin motors for transport (for review see Engqvist-Goldstein and Drubin, 2003; Munn, 2000). Endosomes that have abscised from the plasma membrane have been proposed to move independently of actin polymerization because Las17 and certain actin binding proteins are depleted from endocytic F-actin patches as they internalize from the cell cortex (Kaksonen et al., 2003). In this model, the force of internalization is hypothesized to impart momentum to endosomes, moving them through the cytoplasm. However, whether this mechanism could account for the distance and speed with which endosomes travel through the viscous cytoplasm is unclear.
In contrast, results presented here and elsewhere indicate that Ste2-containing endosomes are transported by ongoing and continual polymerization of actin filaments from their membrane surface. First, the actin polymerization toxins latrunculin A and jasplakinolide rapidly (<5 min) inihibit endosome motility (Chang et al., 2003; unpublished results). Second, endocytic internalization is insufficient to support endosome motility because the WCA domain of Las17 is dispensable for Ste2 internalization whereas it is required for motility of Ste2-containing endosomes (Chang et al., 2003). This conclusion is also supported by results of the present study showing that endosome motility but not endocytic internalization is inhibited when actin filament barbed ends are capped by overexpressing capping protein. Third, attaching the WCA domain of Las17 to the endocytic cargo Ste2 rescues the motility defect of las17WCA cells. Fourth, Ste2-containing endosomes can stop and start, make sharp turns or reverse direction (Chang et al., 2003), indicating that movement is not inertial.
These observations are consistent with two hypotheses. First, Las17 activates the Arp2/3 complex on the plasma membrane, producing endosomes with uncapped actin filaments that continue to polymerize and depolymerize, thereby powering movement. Alternatively, Las17 activates the Arp2/3 complex directly on endosome membranes, thereby stimulating ongoing actin filament assembly that drives motility. Although the Arp2/3 complex, Las17 and F-actin have yet to be localized on Ste2-labeled endosomes, these proteins may be present below the detection limit because endosomes are small and actin filaments are short.
Does yeast have more than one endocytic process?
To integrate our findings with those of previous investigations, we suggest that yeast cells possess at least two endocytic processes distinguished by their internalization sites and mechanisms of endosome transport via the actin cytoskeleon, analogous to the diverse roles of the mammalian actin cytoskeleton in endocytosis (reviewed in Engqvist-Goldstein and Drubin, 2003). One process mediates endocytosis of the GPCR Ste2; whether other cargo use this mechanism remains to be determined. In this mechanism, Ste2 internalizes at small invaginations associated with relatively little actin, as shown by immunoelectron microscopy (Mulholland et al., 1999). As Ste2-containing endosomes form, they continue to nucleate actin filaments by a mechanism requiring Las17 and the Arp2/3 complex (Chang et al., 2003; results of the present investigation), thereby transporting endosomes along irregularly shaped paths to the lysosome-like vacuole. Such nonlinear motility may also allow endocytic cargo to recycle to the plasma membrane, as occurs with the GPCR Ste3 (Davis et al., 1993; Luo and Chang, 2000).
In a second process, endocytic internalization occurs at cortical F-actin patches, as observed using FM 464 or GFP-labeled actin-binding proteins. Here, Pan1, Las17, type I myosins (Myo3 and Myo5), and other proteins activate the Arp2/3 complex (Evangelista et al., 2000; Geli et al., 2000; Lechler et al., 2000; Duncan et al., 2001; Young et al., 2004), thereby nucleating actin filament assembly and endosome scission from the plasma membrane. A subpopulation of the endosomes produced by this mechanism are transported to the vacuole along actin cables that treadmill (Huckaba et al., 2004). This transport process may efficiently couple secretion and endocytosis during polarized cell growth because cortical actin patches colocalize with the ends of actin cables and because actin cables deliver secretory vesicles to cortical sites of cell growth (Pruyne et al., 1998, 2002; Evangelista et al., 2002).
Type II PI 4-kinases in endocytosis and vesicular trafficking
Type II PI 4-kinases are a newly appreciated family of enzymes characterized by catalytic domains dissimilar to those of other PI kinases (Minogue et al., 2001; Wei et al., 2002). Mammalian cells express and ß isoforms of type II PI 4-kinase that are products of distinct genes. Type II PI 4-kinases are peripheral membrane proteins that associate with the plasma membrane, ER, Golgi apparatus, endosomes, synaptic vesicles, and F-actin (Balla et al., 2002; de Graaf et al., 2002; Guo et al., 2003; Wang et al., 2003; Waugh et al., 2003; Carloni et al., 2004). The type II
isoform produces a pool of PI4P that recruits protein scaffolds implicated in cytoskeletal organization and synaptic vesicle budding (Guo et al., 2003), or that regulates the clathrin adaptor AP-1 on Golgi membranes (Wang et al., 2003). Otherwise, the functions of type II PI 4-kinases are poorly understood.
Before our study, Lsb6, the sole type II PI 4-kinase of yeast, had been characterized biochemically (Han et al., 2002; Shelton et al., 2003). However, its function was unknown because lsb6 mutants did not exhibit growth defects (Han et al., 2002; Shelton et al., 2003), in contrast to the lethal growth deficits of mutants defective in the type III PI 4-kinases Stt4 or Pik1. However, we show herein that Lsb6 is required for the motility of endosomes containing Ste2, a novel function for type II PI 4-kinases. Strikingly, we find that the PI 4-kinase activity of Lsb6 is completely dispensable for endosome motility. Instead, Lsb6 promotes endosome motility by interacting directly or indirectly with the WASp homologue Las17, which in turn activates the Arp2/3 complex, resulting in actin filament assembly on endosome membranes. This conclusion is supported by results showing that the NH2-terminal half of Lsb6 is necessary and sufficient for endosome motility and interaction with Las17. The NH2 terminus of Lsb6 is highly conserved among fungi, with two clusters of conserved hydrophobic residues that may play a role in this process (Fig. S4).
Several findings suggest that Lsb6 may promote endosome motility by activating Las17. First, Lsb6 and Las17 localize to plasma and vacuole membranes (Madania et al., 1999; Eitzen et al., 2002). Second, Las17 functions downstream of Lsb6 because appending the WCA domain of Las17 to Ste2 rescues the endosome motility defect of lsb6 mutants. Third, Lsb6 may activate rather than recruit Las17 because Las17 is localized normally in lsb6
mutants (unpublished data). Biochemical studies are underway to determine whether Lsb6 stimulates the ability of Las17 to activate the Arp2/3 complex.
In conclusion, our findings support the hypothesis that endosomes bearing the GPCR Ste2 move by force generated from ongoing polymerization of actin filaments on endosome membranes via the action of the WASP homologue Las17 and the Arp2/3 complex. This mechanism requires a type II PI 4-kinase, Lsb6, functioning independently of its enzymatic activity as an upstream regulator of Las17. Because Lsb6, Las17, and the Arp2/3 complex are highly conserved in eukaryotes, their counterparts in other organisms may have critical roles regulating endocytic transport of many signaling receptors, transporters, or intracellular pathogens.
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Materials and methods |
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PI 4-kinase assays
Cells were grown to exponential phase (OD600 0.5). We grew lsb6
cells in synthetic complete (SC) medium and lsb6
cells containing plasmids in synthetic complete medium lacking histidine and uracil. Cell extracts were prepared as described previously (Han et al., 2002). For immunoblot analysis, cell extracts were resolved by SDS-PAGE using a 10% slab gel, transferred to a PVDF membrane, and subjected to reaction with anti-HA antibodies at a concentration of 1 µg/ml. PI 4-kinase activity was measured for 10 min at 30°C in the presence of 50 mM Tris-maleate (pH 7.0), 2.5 mM [
-32P]ATP (10,000 cpm/nmol), 10 mM MgCl2, 0.2 mM PI, 3.2 mM Triton X-100, and 20 µg of cell extract protein in a total volume of 0.1 ml (these are optimal conditions for Lsb6 kinase activity relative to other PI 4-kinases as described in Han et al. (2002). The 32P-labeled chloroform-soluble products of the reaction were dried and used for scintillation counting. All assays were conducted in triplicate and were linear with time and protein concentration. A unit of PI 4-kinase activity was defined as the amount of enzyme that catalyzed the formation of 1 nmol of product/min. Specific activity was defined as units per mg of protein.
Microscopy
Rhodamine-phalloidin (Invitrogen) and FM 464 (Invitrogen) were used according to the manufacturers' directions. The plasmid pUG34-PLCPH2GFP was used according to published methods (Wild et al., 2004). Yeast cells were grown in synthetic dextrose media overnight, subcultured, and allowed to grow overnight to an OD600 of 0.250.4. Cells were then washed twice and concentrated in nonfluorescent media. For temperature-sensitive mutants, all mutants were grown in synthetic media and switched to nonpermissive temperature for the indicated times, washed twice with nonfluorescent media, and visualized immediately on a heated microscope stage. For capping protein overexpression, cells were grown in synthetic media containing raffinose overnight. Induction was done by adding galactose to a final concentration of 2%, and cells were allowed to induce to 2 h until maximal expression was achieved as indicated by immunoblotting. Cells were mounted on glass slides, covered with a glass coverslip, and viewed with an Olympus IX70 inverted epifluorescence microscope and a cooled CCD camera (RC300; Dage-MTI). Single focal plane images were obtained using U-MNG (rhodamine-phalloidin) and U-MWIBA (GFP) filter sets captured using NIH Image (http://rsb.info.nih.gov/nih-image). Each frame was taken by integrating for 300500 ms; frames were separated by 500700 ms until 50 frames had been acquired. For experiments using temperature-sensitive mutants, a plastic tent was set up such that the microscope system was heated for 23 h before the experiment and maintained at the indicated nonpermissive temperature for the duration of the experiment.
Quantification of endosome motility
Time-lapse images of cells expressing Ste2-GFP were analyzed manually and where indicated by using automated procedures. For manual analysis, four sequential frames from movies were chosen and three endosomes were tracked in the XY plane. Very bright late endosomes near the vacoule or GFP aggregates on the plasma membrane were not scored. As endosomes in wild-type cells are highly motile, four frames in any given movie were sufficient to analyze motility of several endosomes. The number of endosomes per cell was variable as a function of growth conditions (unpublished data). The distance traveled by a given endosome between each time point was calculated based on pixel coordinates (10.2 pixels/micron), which allowed the average speed of each endosome to be calculated. For a given cell type or experimental condition, the average endosome speed, standard error and P value were calculated from data obtained by imaging 3572 endosomes (Table I) using the unpaired Student's t test. A P value < 0.005 when compared with matched wild-type controls was considered significant. For each experiment, three independent transformants were isolated, visualized, and the data quantified. Each experiment was repeated two to four times.
When using an automated particle tracking program to analyze endosome motility (TrackerX; Carlsson et al., 2002), we transferred time-lapse images into text files using ImageJ 1.32g (http://rsb.info.nih.gov/objectimage). Endosomes were tracked for a minimum of 5 s as described previously (Carlsson et al., 2002); data obtained from 60 to 140 endosomes were averaged per experiment. Because 70% of endosomes in wild-type cells moved rapidly and left the plane of focus over a period of 25 s, shorter time courses (1015 s) were used. Conversely, poorly motile endosomes in mutants remained in the plane of focus much longer.
Yeast two hybrid assays
Two hybrid plasmids were introduced into PJ69-4A cells as described by the manufacturer (CLONTECH Laboratories, Inc.). At least three transformants were isolated and assayed for ß-galactosidase activity in extracts using o-nitrophenyl-BD-galactopyranoside (ONPG; Sigma-Aldrich) as substrate. Results shown are the average of at least three independent experiments.
Online supplemental material
Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200501086/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM44592 and HL075632 (to K.J. Blumer) and GM28140 (to G.M. Carman) and by an American Heart Association Predoctoral Fellowship (0215240Z to F.S. Chang).
Submitted: 19 January 2005
Accepted: 2 September 2005
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