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HPS6 interacts with dynactin p150Glued to mediate retrograde trafficking and 1 

maturation of lysosomes 2 

Ke Li1, 2, Lin Yang1, Cheng Zhang1, Yang Niu1, 2, Wei Li1 and Jia-Jia Liu1* 3 

1State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and 4 

Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China 5 

2University of Chinese Academy of Sciences, Beijing 100039, China 6 

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Running title: HPS6 in lysosomal trafficking 9 

Key words: HPS6, p150Glued, lysosome, dynein–dynactin 10 

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*Author for correspondence: Jia-Jia Liu (jjliu@genetics.ac.cn) 12 

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© 2014. Published by The Company of Biologists Ltd.Jo

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JCS Advance Online Article. Posted on 4 September 2014

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Summary 14 

HPS6 was originally identified as a subunit of the BLOC-2 protein complex which is 15 

involved in the biogenesis of lysosome-related organelles (LRO). Here, we 16 

demonstrate that HPS6 directly interacts with the p150Glued subunit of the 17 

dynein–dynactin motor complex and acts as cargo adaptor for the retrograde motor to 18 

mediate transport of lysosomes from the cell periphery to the perinuclear region. 19 

Small interference RNA (siRNA)-mediated knockdown of HPS6 in HeLa cells not 20 

only partially blocks centripetal movement of lysosomes but also causes delay in 21 

lysosome-mediated protein degradation. Moreover, lysosomal acidification and 22 

degradative capacity as well as fusion between LE/MVB and lysosome are also 23 

impaired when HPS6 is depleted, suggesting that dynein–dynactin-mediated 24 

perinuclear positioning is required for lysosome maturation and activity. Our results 25 

have uncovered a novel specific role for HPS6 in the spatial distribution of the 26 

lysosomal compartment. 27 

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Introduction 30 

The intracellular transport driven by motor proteins plays critical roles in membrane 31 

trafficking that is essential for organelle biogenesis, homeostasis and function. 32 

Cytoplasmic dynein, a microtubule-based motor, drives the movement of a wide range 33 

of cargoes towards the minus ends of microtubules and is primarily responsible for 34 

retrograde transport of materials from the cell periphery to the center (Allan, 2011). 35 

Functions of dynein require the activity of dynactin, a multisubunit protein complex 36 

that not only enhances its processivity along microtubule tracks (Culver-Hanlon et al., 37 

2006; King and Schroer, 2000; Moore et al., 2009) but also links it to cargoes. 38 

Although several proteins that tether dynein–dynactin to its membranous cargoes have 39 

been identified and their functions in intracellular trafficking characterized 40 

(Engelender et al., 1997; Hong et al., 2009; Hoogenraad et al., 2001; Johansson et al., 41 

2007; Tai et al., 1999; Traer et al., 2007; Wassmer et al., 2009; Watson et al., 2005; 42 

Yano and Chao, 2005), adaptor proteins for many other organelles and vesicular 43 

cargoes retrogradely transported from the cell periphery to the center remain to be 44 

discovered. 45 

Lysosomes are catabolic organelles of eukaryotic cells that contain hydrolytic 46 

enzymes required for degradation of macromolecules including proteins, lipids, 47 

nucleic acids and polysaccharides. They play a central role in degradation of 48 

macromolecules received from endocytic, phagocytic and autophagic pathways, 49 

which is essential for many cellular and physiological processes such as 50 

receptor-mediated signaling, antigen presentation, pathogen clearance and cholesterol 51 

homeostasis (Saftig and Klumperman, 2009; Settembre et al., 2013). The intracellular 52 

trafficking and steady-state distribution of lysosomes rely upon molecular motors and 53 

both the actin and microtubule cytoskeletons (Burkhardt et al., 1997; Cantalupo et al., 54 

2001; Cordonnier et al., 2001; Harada et al., 1998; Jordens et al., 2001; Matsushita et 55 

al., 2004; Rosa-Ferreira and Munro, 2011; Santama et al., 1998). Although it was 56 

reported that lysosomes change their intracellular positioning to coordinate cellular 57 

nutrient responses through mTOR signalling and autophagic flux (Korolchuk et al., 58 

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2011), the physiological relevance of perinuclear steady-state clustering of lysosomes 59 

in cells remains largely unexplored. 60 

Lysosome-related organelles (LRO) are a class of tissue-specific compartments 61 

that share many features with lysosomes but store and secret proteins for 62 

cell-type-specific functions (Marks et al., 2013; Raposo et al., 2007). They include 63 

melanosomes, platelet dense granules, lytic granules, lamellar bodies and many others 64 

(Raposo et al., 2007). Defects in the biogenesis of these organelles 65 

cause Hermansky–Pudlak syndrome (HPS), a group of human autosomal recessive 66 

disorders characterized by partial loss of pigmentation and prolonged bleeding (Wei 67 

and Li, 2013). Except for the HPS2 gene which encodes the subunit of AP-3 68 

complex, genes mutated in the other eight forms of HPS encode subunits of three 69 

protein complexes named Biogenesis of Lysosome-related Organelle Complex 70 

(BLOC)-1, -2 and -3, with HPS7, 8 and 9 of BLOC-1, HPS3, 5 and 6 of BLOC-2, and 71 

HPS1 and 4 of BLOC-3 (Di Pietro et al., 2004; Gautam et al., 2004; Li et al., 2003; 72 

Wei and Li, 2013; Zhang et al., 2002; Zhang et al., 2003). While it has been 73 

established that the BLOCs are important for LRO biogenesis (Chiang et al., 2003; Di 74 

Pietro et al., 2006; Martina et al., 2003; Setty et al., 2007), and that biological 75 

functions of BLOC-1 and BLOC-3 have been identified most recently 76 

(Gerondopoulos et al., 2012; John Peter et al., 2013; Kloer et al., 2010), the exact 77 

molecular functions of the BLOC-2 subunits remain elusive. 78 

In this study, we found that HPS6 is a cargo adaptor for dynein–dynactin. We 79 

present evidence that HPS6 binds to dynactin p150Glued, and that this interaction is 80 

required for the perinuclear positioning of lysosomes. Depletion of HPS6 in HeLa 81 

cells not only caused mislocalization of LAMP1/2-positive lysosomes to the cell 82 

periphery, but also impaired lysosomal degradation of endocytic cargoes, which could 83 

be rescued by overexpressing full-length HPS6 but not HPS6 truncation mutants that 84 

are unable to bind p150Glued. Further, lysosomal acidification and fusion between late 85 

endosomes (LEs)/multivesicular bodies (MVBs) and lysosomes were also impaired in 86 

HPS6-depleted cells. We propose that HSP6 facilitates retrograde lysosomal 87 

trafficking by linking the retrograde motor to lysosomes and that perinuclear 88 

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positioning of lysosomes is crucial for delivery of endocytic cargoes to lysosomes, 89 

lysosome maturation and functioning. 90 

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Results 91 

HPS6 directly interacts with the p150Glued subunit of the dynein–dynactin 92 

complex 93 

In search of cargo adaptors for the dynein–dynactin motor, we performed a yeast 94 

two-hybrid screen of a human fetal brain cDNA library using the carboxyl 95 

(C)-terminus of human dynactin p150Glued (amino acid residues (aa) 718-1278) as bait 96 

(Hong et al., 2009). One out of 20 positive clones was found to encode an amino 97 

(N)-terminal fragment (aa 66-197) of HPS6. To verify the protein-protein interaction 98 

in mammalian cells, we transiently co-transfected HEK293 cells with plasmids 99 

encoding epitope-tagged full-length p150Glued and HPS6, and performed 100 

co-immunoprecipitation (co-IP) with immobilized anti-Flag antibodies. HPS6 and 101 

p150Glued reciprocally co-immunoprecipitated with each other (Figure 1A), indicating 102 

that the interaction between HPS6 and p150Glued also occurs in mammalian cells. To 103 

further verify the interaction, we performed co-IP with HeLa cell lysates and detected 104 

not only HPS3 and HPS5, the other two subunits of the BLOC-2 complex, but also 105 

p150Glued and dynein intermediate chain (DIC) in the immunoprecipitates of HPS6 106 

(Figure 1B). 107 

To map the HPS6 interaction site on p150Glued, we performed yeast two-hybrid 108 

assays (Figure 1D) and coIP experiments in cultured mammalian cells with a series of 109 

p150Glued fragments. In mammalian cells, although the extreme C-terminus of 110 

p150Glued was not expressed, co-IP of cell extracts co-expressing HPS6 and p150Glued 111 

fragments showed that the C-terminal fragment of aa 911-1281 interacts strongly with 112 

HPS6 (Figure 1E and F). These data together indicate that the HPS6-binding site on 113 

p150Glued lies in its extreme C-terminus. We also attempted to map the p150Glued 114 

interaction site(s) on HPS6 by yeast two-hybrid assays and co-IP of cell extracts 115 

co-expressing p150Glued and a series of HPS6 fragments. Interaction between the 116 

N-terminal region of HPS6 (aa 66-200) and the extreme C-terminus of p150Glued (aa 117 

1138-1281) was verified in the yeast two-hybrid assay (Figure 1G). Although the 118 

extreme N-terminal fragments of HPS6 were not expressed in either yeast or 119 

mammalian cells, surprisingly, all of the N-terminal fragments (aa 1-400, 1-500 and 120 

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1-700) that were expressed in cultured cells failed to co-immunoprecipitate p150Glued 121 

(Figure 1H), suggesting that the region adjacent to the extreme N-terminus might 122 

inhibit its interaction with p150Glued through aberrant protein folding. Intriguingly, 123 

among the truncation mutants expressed, an N-terminally truncated fragment (aa 124 

201-805) was able to bind p150Glued, and another N-terminal truncation fragment (aa 125 

301-805) showed weak p150Glued binding (Figure 1H). Taken together, the finding that 126 

HPS6 interacts with p150Glued prompted us to hypothesize that HPS6 serves as cargo 127 

adaptor to mediate dynein–dynactin-driven retrograde transport. 128 

129 

The perinuclear distribution of HPS6 requires dynein–dynactin activity 130 

Immunofluorescence staining of HeLa cells with antibodies to HPS6 revealed that it 131 

primarily localized to perinuclear vesicular structures (Figure S1). To test whether the 132 

perinuclear distribution of HPS6-associated membranous structures requires 133 

dynein–dynactin-mediated retrograde transport, we treated cells with nocodazole to 134 

disrupt the microtubule cytoskeleton, which serves as track for the retrograde motor. 135 

Indeed, nocodazole treatment resulted in disassembly of the microtubule network and 136 

dispersal of HPS6 signals to the cell periphery (Figure 2A). Moreover, overexpression 137 

of a Flag-tagged p50/dynamitin subunit of dynactin, which disassembles the 138 

dynein–dynactin protein complex (Echeverri et al., 1996; Melkonian et al., 2007), also 139 

caused dispersal of HPS6 signals to the cell periphery (Figure 2B), indicating that the 140 

perinuclear distribution of HPS6-associated vesicles requires both intact microtubule 141 

cytoskeleton and dynein–dynactin activity. 142 

Since p150Glued binds to HPS6 through its C-terminus, and its N-terminal and 143 

middle regions mediate its interaction with DIC and the Arp1 subunit of dynactin, 144 

respectively (Fig. 1C) (Schroer, 2004), we reasoned that in the absence of the 145 

cargo-recognition site, the N-terminal fragment of p150Glued alone could serve as a 146 

dominant negative mutant to compete with the full-length protein in the motor 147 

complex. In agreement with our previous study, in which SNX6-mediated, 148 

dynein–dynactin-driven retrograde transport of CI-MPR from endosomes to the TGN 149 

was disrupted by overexpression of a p150Glued truncation mutant (aa 1-910, 150 

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Flag-p150Glued-N) lacking the SNX6-binding C-terminus (Hong et al., 2009), 151 

overexpression of the same protein fragment perturbed HPS6 distribution, causing its 152 

cytoplasmic dispersal, whereas overexpression of its HPS6-binding C-terminus (aa 153 

911-1281, Flag-p150Glued-C) had no obvious effect (Figure 2B), probably because it 154 

cannot assemble into the dynactin complex by itself and thus cannot compete with the 155 

endogenous, wild-type protein for vesicular cargoes (Johansson et al., 2007). 156 

Therefore, these data establish that the perinuclear distribution of HPS6 requires 157 

dynein–dynactin-driven retrograde transport. 158 

159 

HPS6 associates with the lysosomal compartment 160 

To investigate cellular function(s) of HPS6, first we analyzed its subcellular 161 

compartmental distribution by immunofluorescence staining and confocal microscopy 162 

of HeLa cells. Colocalization analysis revealed that HPS6 partially colocalized with 163 

CD63 (also known as LAMP3), the LE/MVB marker (Figure 3A-B). Intriguingly, 164 

HPS6 colocalized to a higher extent with lysosome-associated membrane proteins 1 165 

and 2 (LAMP1 and LAMP2) (Figure 3A-B), two of the most abundant lysosomal 166 

proteins (Eskelinen, 2006). In contrast, little colocalization between HPS6 and the 167 

early endosome marker EEA1 was observed, neither was the trans-Golgi network 168 

(TGN) resident protein p230 (Figure 3A-B). Moreover, not only p150Glued, but also 169 

LAMP1 and LAMP2 were detected on HPS6-positive vesicles immunoisolated from 170 

membrane fractions of HeLa cells (Figure 3C). Conversely, immunoisolation of 171 

LAMP1-positive vesicles from membrane fractions showed that not only LAMP2, but 172 

also HPS6 and p150Glued associate with LAMP1-positive lysosomes (Figure 3D). To 173 

further determine whether HPS6 is indeed associated with lysosomes, we performed 174 

ultrastructural analysis of immunoisolated HPS6-associated vesicles on magnetic 175 

beads by transmission electron microscopy. Electron-dense lamellar lysosomal 176 

structures were found to be associated with HPS6-conjugated beads, whereas no 177 

vesicular structures in association with IgG-conjugated beads were detected (Figure 178 

3E). In contrast, when immunoisolation was performed with antibodies to GM130, a 179 

cis-Golgi marker, Golgi cisternae-like structures were found associated with 180 

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GM130-conjugated beads (Figure 3E). Together these data indicate that HPS6 181 

associates with lysosomes. 182 

183 

HPS6 mediates minus end-directed microtubule motility and perinuclear 184 

positioning of lysosomes 185 

Having established that HPS6 requires dynein–dynactin for perinuclear distribution 186 

and that HPS6 associates with lysosomes, we reasoned that HPS6 might play a role in 187 

retrograde transport and subcellular distribution of lysosomes. To investigate role(s) 188 

of HPS6 in lysosomal transport and positioning, we depleted HPS6 by 189 

siRNA-mediated RNA interference (Figure S1E-F). Immunofluorescence microscopy 190 

showed that both LAMP1- and LAMP2-labeled lysosomes were evenly distributed in 191 

cells depleted of HPS6 (Figure 4A and B). The HPS6 deficiency-induced subcellular 192 

distribution phenotype of LAMP1 was rescued by Flag-tagged, RNAi-resistant HPS6 193 

(Figure 4D and E), indicating that HPS6 is specifically required for the perinuclear 194 

positioning of lysosomes. Further, overexpression of the HPS6 N-terminal fragment 195 

(aa 1-700), which could not bind to p150Glued but retains the ability to bind to HPS5, 196 

another BLOC-2 subunit (Figure 4C), did not rescue the lysosome dispersal 197 

phenotype (Figure 4D and E), indicating that the p150Glued-binding ability of HPS6 is 198 

required for the perinuclear positioning of lysosomes. Overexpression of Flag-HPS6, 199 

however, did not affect subcellular distribution of LAMP1 lysosomes in HeLa cells 200 

(Figure 4F and H). 201 

Having established that HPS6 is required for lysosome perinuclear positioning 202 

and that HPS6 interacts with the dynein-dynactin motor complex, next we asked 203 

whether HPS6 mediates retrograde transport of lysosomes by linking the motor to 204 

lysosomal cargoes. Since we have demonstrated that overexpression of p150Glued-N 205 

caused cytoplasmic dispersal of HPS6, possibly by competing with endogenous 206 

p150Glued for HPS6 binding, we reasoned that this p150Glued fragment could also block 207 

retrograde transport mediated by HPS6. Indeed, immunofluorescence microscopy 208 

showed that overexpression of Flag-p150Glued-N but not Flag-p150Glued-C caused 209 

dispersal of LAMP1-labeled lysosomes to the cell periphery (Figure 4G-H), 210 

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indicating that the interaction of HPS6 with p150Glued is required for lysosomal 211 

positioning in the perinuclear region. Similarly, consistent with previous studies 212 

(Burkhardt et al., 1997), overexpression of Flag-p50, which disrupts the 213 

dynein–dynactin protein complex, caused peripheral distribution of lysosomes (Figure 214 

4G-H). 215 

To test whether HPS6 is required for the association of dynein–dynactin with 216 

lysosomes, we performed immunoisolation of lysosomes from HeLa cell lysates with 217 

antibodies to LAMP1. Immunoblotting analysis indicated that there was a significant 218 

decrease in the levels of p150Glued as well as DIC co-immunoisolated with LAMP1 in 219 

cells depleted of HPS6 (Figure 4I). It was reported that in the fibroblasts from Hps1 220 

mutant mice (pale ear, BLOC-3 deficient), LAMP1-positive compartments were less 221 

concentrated than wild-type, whereas their perinuclear clustering and surface levels 222 

were normal in fibroblasts from Hps3 mutant mice (cocoa, BLOC-2 deficient) 223 

(Falcon-Perez et al., 2005; Salazar et al., 2006). To determine whether loss of HPS6 224 

function in mouse causes similar phenotype of LAMP1 mislocalization to that caused 225 

by HPS6 gene knockdown, we performed immunofluorescence staining of MEF from 226 

ruby-eye (ru) mouse, which harbors an internal deletion (aa 187-189) mutant of HPS6 227 

that cannot interact with p150Glued (Figure 5A). Confocal microscopy analysis showed 228 

that compared to wild-type (WT) MEF, both LAMP1 and LAMP2 were more 229 

peripherally distributed in ru MEF (Figure 5B), indicating that loss of HPS6 function 230 

indeed leads to cytoplasmic dispersal of lysosomes. Consistently, immunoisolation 231 

from brain lysates of ru mouse with antibodies to LAMP1 detected less p150Glued and 232 

DIC on LAMP1 vesicles than from that of wild-type mouse (Figure 5C). Moreover, 233 

the cytoplasmic dispersal phenotype of LAMP1 in ru MEF was also rescued by the 234 

full-length HPS6 fused to EGFP (Figure 5D). 235 

To further verify that HPS6 mediates retrograde transport of lysosomes driven by 236 

dynein-dynactin, we performed live imaging experiments. Image analyses of 237 

trajectories of mCherry-LAMP1-labeled vesicles indicate that, compared with control 238 

cells, indeed there was a significant decrease in movement of LAMP1 vesicles 239 

towards the cell center in HPS6-depleted cells (Fig. S2). Taken together, these results 240 

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indicate that the perinuclear positioning of lysosomes requires 241 

dynein–dynactin-driven retrograde transport and that HPS6-p150Glued interaction 242 

mediates the retrograde transport of lysosomes to the cell center. 243 

That HPS6 is required for perinuclear distribution of lysosomes prompted us to 244 

ask whether it is also required for subcellular distribution of late endosomes and 245 

MVBs. Immunofluorescence microscopy of HeLa cells with antibodies to Rab7 and 246 

CD63, markers for LEs and MVBs, respectively, did not detect any changes in the 247 

distribution pattern of Rab7 and CD63-positive structures in HPS6-depleted cells 248 

(Figure S3A-D). Moreover, no change was detected in the subcellular distribution of 249 

CD-MPR and CI-MPR (Figure S3E-H), carrier proteins for lysosomal hydrolases that 250 

shuttle between the TGN and endosomes (Diaz and Pfeffer, 1998; Saftig and 251 

Klumperman, 2009; Seaman, 2004). Thus, HPS6 is not required for LE/MVB 252 

positioning and trafficking between endosomes and the TGN. 253 

Previously it was reported that RILP binds to p150Glued and mediates retrograde 254 

transport of lysosomes towards the cell center (Johansson et al., 2007). To test 255 

whether there is functional redundancy between HPS6 and RILP in lysosomal 256 

movement and positioning, we performed double knockdown of HPS6 and RILP and 257 

examined lysosome distribution by immunostaining followed by quantitative analysis. 258 

Radial profile plots showed that lysosomes were more peripherally distributed in 259 

RILP- or HPS6-depleted cells, and double knockdown caused further shift of LAMP1 260 

fluorescence towards the cell periphery (Fig. S4A-C), suggesting that partially 261 

redundant or compensatory mechanisms exist to mediate centripetal movement and 262 

perinuclear positioning of lysosomes. Moreover, we examined HPS6 colocolization 263 

with RILP-labeled lysosomes and found that, consistent with previous reports 264 

(Cantalupo et al., 2001; Jordens et al., 2001), RILP colocalized with LAMP1 and 265 

Rab7 (Fig. S4D). In contrast, RILP did not colocalize with HPS6 (Fig. S4D), 266 

suggesting that they mediate different retrograde lysosomal transport pathways. 267 

268 

HPS6 and its interaction with p150Glued are required for lysosomal degradation 269 

of endocytosed cargoes and lysosomal enzyme activity 270 

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Previous studies have found that lysosomal positioning coordinates nutrient responses 271 

through regulation of mTORC1 signaling and autophagic flux (Korolchuk et al., 272 

2011). To assess the physiological relevance of HPS6-mediated perinuclear 273 

positioning of lysosomes, first we examined lysosomal activity in HPS6-depleted 274 

cells. To this end, we incubated HeLa cells with DQ-BSA and measured its 275 

fluorescence intensity, which is an indication of lysosomal degradation capacity as 276 

endocytosed DQ-BSA became fluorescent upon proteolytic cleavage in lysosomes 277 

(Vazquez and Colombo, 2009). Compared with control cells, weaker DQ-BSA 278 

fluorescence was detected in HPS6-depleted cells up until 3 hours of incubation 279 

(Figure 6A-B), indicating that HPS6 depletion delays the degradation of endocytosed 280 

cargo in lysosomes. Similarly, when we incubated cells with Magic Red cathepsin B 281 

(MR catB) substrate, a membrane permeable probe that fluoresces upon cleavage by 282 

the lysosomal protease cathepsin B (Creasy et al., 2007a), a decrease in the mean 283 

intensity of fluorescent puncta was detected in HPS6-depleted cells (Figure 6C-D), 284 

indicating that lysosomal enzyme activity was impaired. Overexpression of 285 

RNAi-resistant full-length HPS6 restored lysosomal degradation of DQ-BSA and MR 286 

catB, whereas the truncation mutant which failed to interact with p150Glued but 287 

retained HPS5-binding ability had no effect (Fig. 6E-H). Moreover, overexpression of 288 

EGFP fusion proteins of either p50 or p150Glued-N, but not p150Glued-C, caused a 289 

decrease in the fluorescence intensity of MR catB substrate puncta (Figure 6I-J). 290 

Together these data indicate that HPS6 and its interaction with p150Glued are required 291 

for both degradation of endocytic cargo and enzyme activity of lysosomes. 292 

293 

HPS6 depletion and disruption of dynein-dynactin activity impair fusion 294 

between LE/MVBs and lysosomes 295 

Direct fusion between LE/MVBs and lysosomes was observed and is required for 296 

delivery of endocytic cargoes to lysosomes for degradation (Bright et al., 2005; Bright 297 

et al., 1997; Futter et al., 1996; Mullock et al., 1998). To determine whether the 298 

decreased degradation of endocytic cargo in HPS6-depleted cells is caused by failure 299 

in fusion between LE/MVBs and lysosomes, we labeled lysosomes by incubation of 300 

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cells in medium containing dextran conjugated to Alexa Fluor® 647 (Dextran–647) 301 

for 12 h followed by an 8 h chase in conjugate-free medium. Cells were then 302 

incubated in medium containing dextran conjugated to Alexa Fluor® 488 303 

(Dextran–488) for 3 h followed by confocal imaging (Bright et al., 2005). Confocal 304 

microscopy analysis showed that no difference in the mean intensity and number of 305 

fluorescent puncta was detected (Figure 7A and B), indicating that HPS6 knockdown 306 

did not affect the uptake of dextran. However, there was a decrease in colocalization 307 

between Dextran–488 and Dextran–647 in HPS6-depleted cells (Figure 7A and B), 308 

suggesting that fusion between LE/MVBs and lysosomes requires perinuclear 309 

distribution of lysosomes. Similarly, less Dextran–488 colocalized with 310 

mCherry–LAMP1 in HPS6-depleted cells (Figure 7C and D), indicating that there 311 

was less fusion between LE/MVBs and lysosomes. Further, ultrastructural analysis of 312 

HPS6-depleted cells revealed that, although no significant difference in the size of 313 

lysosomes was detected (Fig. S3I), compared with control cells, there was an increase 314 

in the number of MVBs and a decrease in the number of lamellar-structured and 315 

electron-dense lysosomes (Figure 7E and F). Consistently, overexpression of mCherry 316 

fusion proteins of either p50 or p150Glued-N, but not p150Glued-C, caused a decrease in 317 

colocalization between Dextran–488 and Dextran–647 (Figure 7G and H). Together 318 

these data indicate that fusion between endocytic cargoes and lysosomes requires 319 

HPS6 and dynein-dynactin activity. 320 

321 

Lysosomes fuse with LE/MVBs and mature in the perinuclear region 322 

That HPS6 knockdown caused both cytoplasmic dispersal of lysosomes and impaired 323 

delivery of endocytic cargoes to lysosomes prompted us to speculate that fusion 324 

between LE/MVB and lysosome takes place in the perinuclear region. In support of 325 

this notion, when we plotted the extent of colocalization between Dextran–488 and 326 

Dextran–647 as a function of radial distance starting from the center of the nucleus 327 

determined by its measured centroid (Niu et al., 2013), the radial profile plot revealed 328 

that the extent of colocalization between Dextran–488 and Dextran–647 peaked near 329 

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the nucleus in both control and HPS6-depleted cells (Figure 8A), as well as in cells 330 

overexpressing p50 or p150Glued-N (Figure 8B). 331 

Similarly, we investigated the relationship between lysosomal enzyme activity 332 

and subcellular distribution with radial profile plots of the fluorescent signals of MR 333 

catB substrate. The mean intensity of MR catB fluorescence peaked near the nucleus 334 

not only in control knockdown and HPS6-depleted cells (Figure 8C), but also in cells 335 

overexpressing p50 or p150Glued-N (Figure 8D), indicating that lysosomal enzyme 336 

activity is the highest in the perinuclear region. Since enzymatic activity of lysosomal 337 

proteases is dependent on the pH of lysosomes and there is evidence that lysosomal 338 

acidification is dynamic and tightly regulated (Majumdar et al., 2011; Majumdar et al., 339 

2007; Mindell, 2012), we speculated that lysosomal acidification might also take 340 

place in the perinuclear region. To this end, first we incubated cells overexpressing 341 

mCherry–LAMP1 with LysoSensor (LysoSensorTM green DND-189), a membrane 342 

permeable pH probe (pKa = 5.2) that accumulates and fluoresces green in acidified 343 

compartments within the pH range of 4-5 (Lin et al., 2001; Siemasko et al., 1998). 344 

Intriguingly, compared with control cells, there was a decrease in both the intensity of 345 

LysoSensor fluorescent puncta and its colocalization with mCherry–LAMP1 in 346 

HPS6-depleted cells (Figure 8E and F), indicating that HPS6 knockdown also 347 

impaired lysosome acidification, which contributed to the lower lysosomal enzyme 348 

activity detected with MR catB substrate (Figure 6C-D). Further, the radial profile 349 

plots of LysoSensor showed that the mean intensity of its fluorescence peaked near 350 

the nucleus in both control and HPS6-depleted cells (Figure 8G), indicating that there 351 

is also a positive correlation between lysosomal acidification and perinuclear 352 

positioning. Together these data suggest that the events of heterotypic fusion between 353 

LE/MVBs and lysosomes as well as lysosome acidification and maturation most 354 

frequently take place in the perinuclear region. 355 

That lysosomes fuse with endocytic cargoes and mature in the perinuclear region 356 

prompted us to speculate that fusion of lysosomes with LE/MVBs might play a role in 357 

lysosomal maturation and/or acidification. It was reported that Rab7 is required for 358 

transfer of endocytic cargo from the LE/MVBs to the lysosome (Vanlandingham and 359 

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Ceresa, 2009), probably by regulating the fusion competence of the LE/MVBs. To 360 

determine whether fusion between LE/MVB and lysosome is required for lysosomal 361 

maturation and acquisition of enzyme activity, we depleted Rab7 in HeLa cells by 362 

siRNA knockdown (Figure 8H) and detected lysosomal degradative capacity with MR 363 

catB fluorescence. Compared to control cells, there was a significant decrease in MR 364 

catB fluorescence in Rab7-depleted cells (Figure 8I and J), indicating that indeed 365 

Rab7-mediated fusion between LE/MVBs and lysosomes plays an important role in 366 

lysosomal maturation. 367 

368 

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Discussion 369 

In this study, we started out searching for interaction partners for the p150Glued subunit 370 

of dynein–dynactin and fished out HPS6, a subunit of the BLOC-2 complex involved 371 

in LRO biogenesis and Hermansky–Pudlak syndrome. In HeLa cells, HPS6 is largely 372 

absent from early endosomes and the TGN, and knockdown of HPS6 has no effect on 373 

the distribution of these organelles, neither does it affect the distribution of LEs and 374 

MVBs. Moreover, HPS6 depletion has no effect on endocytic trafficking or trafficking 375 

between endosomes and the TGN. In contrast, while disruption of dynein–dynactin 376 

activity results in block of retrograde transport and peripheral distribution of 377 

lysosomes, HPS6 depletion causes cytoplasmic dispersal of lysosomes, and disruption 378 

of HPS6-p150Glued interaction with the dominant negative p150Glued N-terminus has 379 

similar effect, indicating that HPS6 is specifically involved in dynein–dynactin-driven 380 

retrograde transport of lysosomes towards the cell center (Figure 8K). 381 

It has been found that lysosomes are dispersed in LAMP1/2-deficient cells 382 

(Huynh et al., 2007) and that dynein–dynactin is required for the centripetal 383 

movement of lysosomes (Burkhardt et al., 1997; Cantalupo et al., 2001; Harada et al., 384 

1998; Jordens et al., 2001). However, mechanisms by which the retrograde motor is 385 

recruited to lysosomes are partially understood. Previous studies have identified 386 

multiple dynein recruitment and regulatory factors, including RILP (Cantalupo et al., 387 

2001; Johansson et al., 2007; Jordens et al., 2001) and LIC1/2 (Tan et al., 2011), 388 

dynein cargo adaptors for LEs and lysosomes in HeLa cells. Moreover, it was 389 

reported that in murine fibroblasts deficient in HPS1 (pale ear), not only the 390 

perinuclear clustering of LAMP1-positive LEs and lysosomes but also the frequency 391 

of the movement of LAMP1-GFP-labeled organelles along microtubules was reduced, 392 

suggesting that BLOC-3 might regulate the attachment of LEs and lysosomes to 393 

microtubule motors (Falcon-Perez et al., 2005; Nazarian et al., 2003). In this study, 394 

we identified HPS6 as yet another dynein cargo adaptor for lysosomes that is required 395 

for lysosome perinuclear positioning. Conceivably, these results indicate that multiple 396 

dynein–dynactin recruitment mechanisms exist for lysosome trafficking, which 397 

explains the mild phenotypes caused by HPS6 depletion. Alternatively, different 398 

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dynein adaptors for LE/lysosome and/or LROs might provide cargo 399 

specificity/selectivity that is tissue or cell type-specific and/or organelle specific. 400 

Most recently, BLOC-3 was identified as a guanine nucleotide exchange factor 401 

(GEF) for Rab32 and Rab38, two small GTPases required for melanosome biogenesis 402 

(Gerondopoulos et al., 2012). BLOC-1 was shown to promote endosomal maturation 403 

by recruiting the Rab5 GTPase-activating protein (GAP) Msb3 to endosomal 404 

membrane in yeast (John Peter et al., 2013). However, molecular function(s) of 405 

BLOC-2 subunits remains elusive. Our study on HPS6 reveals its function in 406 

mediating lysosomal trafficking and functioning in the HeLa epithelial cell line, 407 

which is illustrated in the model in Figure 8. It remains to be elucidated whether 408 

HPS6, as a subunit of the BLOC-2 complex, also serves as dynein adaptor in the 409 

biogenesis and trafficking of lysosomes as well as LROs in specialized cell types such 410 

as fibroblasts and melanocytes. 411 

Fusion between LE/MVBs and lysosomes is a critical step in lysosomal 412 

biogenesis and function. Previous studies have demonstrated that Rab7 is required for 413 

efficient fusion between LE/MVBs and lysosomes in HeLa cells, probably through its 414 

interaction with the HOPS (homotypic fusion and vacuole protein sorting) complex 415 

(Luzio et al., 2007; Vanlandingham and Ceresa, 2009). In our study, fusion between 416 

LE/MVBs and lysosomes is impaired in cells depleted of HPS6, accompanying the 417 

more peripheral distribution of LAMP1/2-postivie compartments, suggesting that the 418 

perinuclear clustering of lysosomes is required for proper fusion between LE/MVBs 419 

and lysosomes (Figure 8). Further, blocking LE/MVB-lysosome fusion by Rab7 420 

knockdown also causes a reduction in lysosomal degradative capacity, supporting the 421 

notion that fusion between LE/MVBs and lysosomes to form endo-lysosomes is 422 

required for full maturation and activation of the lysosomal compartment. Previous 423 

studies on lysosomal degradation of amyloid A in microglial cells have provided 424 

strong evidence suggesting that lysosomal acidification is tightly regulated 425 

(Majumdar et al., 2011; Majumdar et al., 2008; Majumdar et al., 2007). It will thus be 426 

important to investigate mechanism(s) by which heterotypic fusion between 427 

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LE/MVBs and lysosomes triggers further acidification and enzyme activation in the 428 

lysosomal compartment. 429 

430 

431 

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Materials and Methods 432 

Antibodies and reagents 433 

Rabbit polyclonal antibodies against HPS6 were raised as described previously 434 

(Gautam et al., 2004). Rabbit polyclonal antibodies against human HPS5 were raised 435 

using recombinant His-HPS5 fragment (aa1-333) purified from E.coli. Antibodies 436 

were affinity purified with the antigen immobilized on AminoLinkagarose gel beads 437 

(Affi-Gel; Bio-Rad Laboratories, Hercules, CA, USA). Other antibodies used in this 438 

study include: mouse anti-p150Glued, mouse anti-EEA1, mouse anti-p230, mouse 439 

anti-CD63, mouse anti-GM130 (BD Biosciences, Mississauga, ON, Canada); mouse 440 

anti-CI-MPR (AbDSeroTec, Oxford, UK); mouse anti-CD-MPR (22d4), mouse 441 

anti-LAMP1 (H4A3), mouse anti-LAMP2 (H4B4) (the Developmental Studies 442 

Hybridoma Bank, Iowa City, IA,USA); mouse anti--actin, rabbit anti-Flag and rabbit 443 

anti-LAMP1 (Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-SNX1, goat 444 

anti-HPS3 (Abcam, CA, USA), goat anti-cathepsin D (R&D,MN, USA), mouse 445 

anti-DIC, rabbit anti-Rab7,goat anti-SNX6, goat anti-cathepsin B, and mouse 446 

anti-c-Myc (Santa Cruz Biotech, Santa Cruz, CA, USA).Fluorescent conjugates of 447 

secondary antibodies andAlexa Fluor 488® phalloidin were purchased from 448 

Invitrogen (Carlsbad, CA, USA), and horse radish peroxidase (HRP)-conjugated 449 

secondary antibodies were from Cell Signaling Technology (Mississauga, ON, 450 

Canada). Sulfo-NHS-LC-Biotin and Streptavidin Agarose Resins were from Thermo 451 

Scientific (Waltham, MA, USA). 452 

453 

Constructs 454 

HPS6 full-length cDNA and fragments were PCR-amplified from the 455 

pGBKT7-mHPS6 construct, and cloned into pCMV-Tag2b and pCMV-Tag3b 456 

(Stratagene, La Jolla, USA) for co-immunoprecipitation assay, or cloned into 457 

pmCherry-C1 and pEGFP-C1 (Clontech, CA, USA) for other experimental purposes. 458 

The p150Glued fragments were PCR-amplified from the Flag-mp150Glued plasmid and 459 

cloned into pCMV-Tag2b (Stratagene, La Jolla, USA) and pEGFP-C2 (Clontech, CA, 460 

USA). The p50 full-length cDNA was PCR-amplified from the Flag-p50 construct 461 

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and cloned into pmCherry-C1 and pEGFP-C2. The mCherry-LAMP1 and myc-RILP 462 

constructs were generous gifts from L. Yu (Tsinghua University, China) and T. Wang 463 

(Xiamen University, China), respectively. 464 

Mouse colonies and cells 465 

The ruby-eye (ru) mutant colonies and the wild-type C57BL/6J were originally from 466 

the Jackson Laboratory and maintained in Dr. Richard Swank’s laboratory at Roswell 467 

Park Cancer Institute (Buffalo, New York, USA) and at the animal facility of the 468 

Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of 469 

Sciences. All procedures were approved by the Institutional Animal Care and Use 470 

Committee of IGDB. The mouse embryonic fibroblasts (MEFs) were isolated from 471 

these mice by following a standard protocol. 472 

473 

Cell culture, siRNA and transfection 474 

HeLa , HEK293 or MEF cells were grown in DMEM (HyClone, Logan, UT, USA) 475 

supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 2 mM 476 

L-glutamine at 37°C with 5% CO2. For DNA transfection, cells were grown to 50% 477 

confluency and transfected with plasmid DNA using LipofectamineTM 2000 478 

(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. For 479 

siRNA transfection, HeLa cells seeded on day 0 were transfected with 50 nM siRNA 480 

duplex using LipofectamineTM 2000 following the manufacturer’s instructions on day 481 

1, seeded and transfected again on day 2, experiments were performed 72 h 482 

post-transfection. For Rab7 knockdown, a mixture of oligonucleotide duplexes 483 

(Vanlandingham and Ceresa, 2009) were transfected as previously described. For 484 

rescue experiments, cells were transfected with plasmid DNA using LipofectamineTM 485 

2000 on day 3 after siRNA transfection, and analyzed for the experiment on day 4. 486 

siRNA sequences used in this study were: 5’-GAGGCUUAGAACAGCUGAATT-3’ 487 

(HPS6) and 5’-UUCUCCGAACGUGUCACGUTT-3’ (non-targeting control). Rab7 488 

siRNA sequences were as follows: 5’-CUAGAUAGCUGGAGAGAUGUU-3’ (#1), 489 

5’-GUACAAAGCCACAAUAGGAUU-3’ (#2), 490 

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5′-AAACGGAGGUGGAGCUGUAUU-3′ (#3), 491 

5′-CGAAUUUCCUGAACCUAUCUU-3′ (#4). 492 

493 

Immunoprecipitation 494 

Immunoprecipitation experiments were performed as previously described (Hong et 495 

al., 2009). For immunoprecipitation assay, HeLa or HEK293 cells were washed once 496 

with PBS and lysed either with lysis buffer A (0.05% [vol/vol] NP-40, 20 497 

mMTris-HCl, pH 7.4, 50 mMNaCl, protease inhibitors) for endogenous IP or with 498 

lysis buffer B (1% [vol/vol] Triton X-100, 50 mMTris-HCl, pH 7.4, 150 mMNaCl, 499 

1mM EDTA and protease inhibitors) for all other IPs. 500 

501 

Immunofluorescence staining and confocal microscopy 502 

Cells were grown on coverslips, fixed in 4% paraformaldehyde/PBS (pH 7.4), and 503 

permeabilized with either PBS containing 0.1% saponin (Sigma-Aldrich, St.Louis, 504 

MO, USA), 1% goat serum, 1% BSA for LAMP1, LAMP2 and CD63 staining or PBS 505 

containing 0.3% Triton X-100, 1% BSA for others. Cells were incubated with primary 506 

antibodies for 1 h at room temperature, and secondary antibodies conjugated with 507 

Alexa Fluor®488, Alexa Fluor® 555 or Alexa Fluor®647 were used for detection. 508 

Images were taken with the Confocal Microscope DIGITAL ECLIPSE C1Si (Nikon, 509 

Japan). 510 

511 

Yeast two-hybrid assay 512 

Matchmaker Gal4 two-hybrid system 3 (Clontech, Mountain View, CA, USA) was 513 

used as previously described (Hong et al., 2009). Yeast strain AH109 was 514 

transformed with pGBKT7 and pGADT7 vectors and plated on tryptophan- and 515 

leucine-deficient SD medium (SD-Leu-Trp). The transformed colonies were further 516 

plated on tryptophan-, leucine-, histidine-, and adenine-deficient plates 517 

(SD-Leu-Trp-Ade-His) for interaction assay. The interaction was assessed by 518 

comparing the growth and blue color of colonies on SD-Leu-Trp-Ade-His containing 519 

2 mg/ml X--Gal. 520 

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521 

Immunoisolation 522 

HeLa cells were homogenized in buffer (10 mMHEPES,pH 7.4, 1 mM EDTA, 0.25 M 523 

sucrose, and protease inhibitors) and centrifuged at 800 × g for 10 min to collect the 524 

supernatant. The supernatant was pooled with the supernatant from previous 525 

centrifugation and subjected to high-speed centrifugation at 100,000 × g at 4°C for 1h 526 

(TLS-55 rotor, OptimaTM MAX Ultracentrifuge; Beckman Coulter, Germany). The 527 

pellet was resuspended in homogenization buffer (membrane fraction) and subjected 528 

to immunoisolation with Dynabeads Protein G (Invitrogen, Carlsbad, CA, USA) 529 

coupled with specific antibodies or control IgG as previously described (Niu et al., 530 

2013). Beads were washed four times with 5% BSA in PBS, and once with 0.1% BSA 531 

in PBS before elution. For detection of organelle markers by immunoblotting, the 532 

beads were eluted by boiling in 2 × SDS gel loading buffer and samples were 533 

analyzed by SDS-PAGE and immunoblotting. For electron microscopy, beads were 534 

processed as follows. 535 

536 

Electron Microscopy 537 

Immunoisolated bead-bound vesicles were fixed with 2.5% glutaraldehyde 538 

(Electronic Microscope Sciences, Hatfield, PA, USA), washed three times with 0.1M 539 

PB buffer (pH 7.4), post-fixed with 1% OsO4, dehydrated via ascending ethanol series, 540 

infiltrated with acetone, and embedded in Embed 812 (Electronic Microscope 541 

Sciences, Hatfield, PA, USA). 542 

The ultrastructural analysis of cells was performed as described (Vanlandingham 543 

and Ceresa, 2009). HeLa cells were transfected with control or HPS6 siRNA and 72 h 544 

post-transfection, cells were washed once with ice-cold PBS and fixed in 2.5% 545 

glutaraldehyde (Electronic Microscope Sciences, Hatfield, PA, USA), 0.2% tannic 546 

acid (Electronic Microscope Sciences, Hatfield, PA, USA), and 2.5% sucrose in 0.1 M 547 

PB buffer (pH 7.4) at 4°C for 2 h, washed three times with 0.1M PB buffer (pH7.4), 548 

scraped, and pelleted. Cells were then post-fixed with 1% OsO4 (SPI Supplies, West 549 

Chester, OH, USA) in 0.1M PB for 1.5 h at 4°C. Samples were then dehydrated in 550 

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ascending acetone series (30%, 50%, 70%, 90%, 5min per dilution, and 100% three 551 

times every 5min) and embedded in Embed 812. 552 

Ultrathin sections (65 nm) were cut with a DiATOME diamond knife (Biel, 553 

Switzerland) on a Leica ultramicrotome (Wien, Austria) and stained with 2% uranyl 554 

acetate and lead citrate. Images were acquired on JEOL JEM-1400 (Tokyo, Japan) 555 

transmission electron microscope equipped with 4K × 2.7K pixels Gatan-832 CCD 556 

camera (Warrendale, PA, USA). 557 

558 

Live cell imaging 559 

Dextran internalization assay was performed as previously described (Bright et al., 560 

2005). Cells were loaded with 0.2 mg/ml Alexa Fluor® 647-conjugated dextran 561 

(10,000 MW, Invitrogen, Carlsbad, CA, USA) at 37°C for 8 h and incubated in 562 

conjugate-free medium for 12 h. The cells were then incubated with 0.5 mg/ml Alexa 563 

Fluor® 488-conjugated dextran (10,000 MW, Invitrogen, Carlsbad, CA, USA) for 3 h, 564 

rinsed with dextran free medium and processed for imaging using Confocal 565 

Microscope DIGITAL ECLIPSE C1Si (Nikon, Japan). 566 

To test lysosomal activity, cells transfected with control or HPS6 siRNA for 48 h 567 

were seeded in 4-well coverglass chambers (Thermo Fisher Scientific, Waltham, MA, 568 

USA). After 16 h, cells were incubated for 1 to 6 h with 0.05 mg/ml DQ-BSA Green 569 

(Invitrogen) (Vazquez and Colombo, 2009), or 2 h with Magic red Cathepsin B 570 

substrate (MR catB, ImmunoChemistry Technology, Bloomington, MN, USA) 571 

(Creasy et al., 2007b) following manufacturer’s instructions. In brief, 26X MR catB 572 

solution was added directly to culture medium at a ratio of 1:26, and then the overlay 573 

cell medium was gently mixed to ensure even exposure to MR catB. After incubating 574 

for 2 h at 37°C, culture medium containing MR catB was removed, and cells were 575 

rinsed twice with PBS. To inhibit the lysosomal V-ATPase activity, 1 M 576 

Bafilomycin A1 (Sigma-Aldrich, St.Louis, MO, USA) was added to the cells for 2 h. 577 

Live cells were stained with Hoechst 33342 for 30 min and imaged as described 578 

above. To test cathepsin B activity in cells overexpressing p50 or p150Glued fragments, 579 

cells transfected with construct expressing EGFP, EGFP-p50, EGFP-p150 Glued-N, or 580 

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EGFP- p150 Glued-C were incubated with MR catB for 2 h as described above, and 581 

imaged by Confocal Microscope DIGITAL ECLIPSE C1Si. 582 

To examine lysosome movement in HeLa cells, control or HPS6 knockdown cells 583 

seeded in 4-well coverglass chambers were transfected with mCherry-LAMP1 for 24 584 

h and stained with Hoechst 33342 for 30 min. Time-lapse fluorescence images were 585 

acquired using DeltaVision OMX V4.0TMSysterms (Applied Precision, USA) with the 586 

following settings: 11 sections; spacing: 0.25 μm; time interval: 4 s; total duration: ~5 587 

min. 588 

589 

Image analysis 590 

Single slice confocal images were analyzed using NIS-Elements AR 3.1 (Nikon, 591 

Japan). For quantification of protein signals on the same immunoblot, images were 592 

analyzed using NIH ImageJ. For quantification of signals in confocal images, images 593 

were subtracted the random fluorescence intensity, which were averaged over 594 

multiple cells. 595 

Blinded quantitative analysis of fluorescent signal distribution was performed on 596 

702 × 702 confocal images using the NIH ImageJ plugin Radial profile as described 597 

(Sengupta et al., 2009). The centroid of the nuclear fluorescent signal was defined as 598 

the center of the concentric circles and it was determined using the ‘Measure’ 599 

function of NIH ImageJ. For each cell, the algorithm measured the mean signal 600 

intensity in a series of concentric circles drawn from the calculated centroid. Average 601 

values from cells over three experiments were then used to generate radial profile 602 

plots in which the fraction of total mean fluorescence is expressed as a function of 603 

radial distance. For quantification of the distribution of signal overlap between 604 

Dextran-488 and Dextran-647, the overlapped fluorescent signals were generated 605 

using the ‘Build Coloc Channel’ function of Imaris version 7.6.5 (Bitplane, Zurich, 606 

Switzerland). To measure the radius of nucleus, at first the freehand selection was 607 

used to trace (or draw) the outline of Hoechst 33342 signal, and then the radial profile 608 

plug-in of NIH ImageJ was used to generate a circle exactly including the Hoechst 609 

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33342 signal and the value of nuclear radius is calculated automatically. The mean 610 

value of nuclear radius was generated from all cells. 611 

The trajectories of mcherry-LAMP1 vesicles were analyzed basically as 612 

previously described (Schuh, 2011). Nucleus and vesicles were detected with the “cell 613 

function” of Imaris and spots were tracked in three dimensions over time. To ensure 614 

that vesicles could be tracked long enough to determine the overall directionality of 615 

their movement, vesicles with displacement length more than 0.35 μm were evaluated. 616 

“Vesicle distance to the closest nucleus center” at each time point was automatically 617 

calculated by the “cell function” and exported into Excel. The direction of vesicle 618 

movement was determined by comparing the beginning and final distance relative to 619 

the center of the nucleus. Since fluorescent signals in live cells photobleached fast 620 

during imaging, only the first 30 images captured in each live imaging experiment 621 

were analyzed. 622 

Quantification of EM images measuring the number of lysosomes, MVBs and 623 

endo-lysosomes per cell profile were done in >100 randomly selected cell profiles 624 

obtained from multiple different grids (> 3) containing ultrathin sections. Structures 625 

of lysosome, MVB, and endo-lysosome in EM images were identified according to 626 

previously reports (Huotari and Helenius, 2011; Luzio et al., 2007; Saftig and 627 

Klumperman, 2009). In brief, lysosomes have an electron-dense lumen with a 628 

diameter ranging from 100 to 1200 nm or lamellar structures with partial electron 629 

dense areas; MVB contain numerous intraluminal vesicles (ILVs) with a diameter of 630 

about 50–100 nm. 631 

Statistical analysis 632 

Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, La 633 

Jolla, CA, USA). Pairwise significance was calculated using two-tailed unpaired 634 

Student’s t test or nonparametric Student’s t test (Mann-Whitney test). For evaluation 635 

of statistical significance of three or more groups of samples, one-way analysis of 636 

variance with a Tukeypost test was used. Results are described as mean ± s.e.m.. Data 637 

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were considered statistically significant once P < 0.05. *P < 0.05; **P < 0.01; ***P < 638 

0.001. 639 

640 

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Acknowledgements 641 

We are indebted to our colleagues for providing reagents (Dr. Li Yu, Tsinghua 642 

University and Dr. Tuanlao Wang, Xiamen University) and for critical comments on 643 

the manuscript (Dr. Shilai Bao, IGDB, CAS). We thank Ling Zhu for technical 644 

assistance and Dr. Zhen-Hua Hao for the preparation of MEF cells and tissues from 645 

wild-type and ruby-eye C57Bl/6J mice. This work was supported by grants from 646 

Ministry of Science and Technology (2011CB965002, 2014CB942802) and the 647 

National Natural Science Foundation of China (31325017 and 31071175 to J-J. Liu, 648 

31230046 to W. Li). 649 

650 

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concurrent fusion and kissing events in living cells. Curr Biol 15, 360‐5. 654 

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dynamitin  (p50)  subunit  of  the  dynactin  complex  disrupts  dynein‐dependent  maintenance  of 659 

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Figure legends 834 

Fig. 1. HPS6 directly interacts with p150Glued. (A) HEK293 cells overexpressing 835 

Flag-tagged p150Glued and myc-tagged HPS6, SNX6 or SNX1 were lysed for co-IP 836 

with immobilized Flag antibody (left). Conversely, HEK293 cells overexpressing 837 

Flag-tagged HPS6 and myc-tagged p150Glued, HPS5 or SNX6 were lysed for co-IP 838 

with immobilized Flag antibody (right). Input and bound proteins were analyzed by 839 

SDS-PAGE and immunoblotting with antibodies against Myc and Flag. (B) Co-IP of 840 

endogenous p150Glued and DIC from HeLa cell lysates with antibodies against HPS6. 841 

Normal IgG and antibodies to SNX1 serve as controls. Input and bound proteins were 842 

analyzed by SDS-PAGE and immunoblotting with antibodies against p150Glued, DIC, 843 

HPS6, HPS3, HPS5, SNX1 and SNX6. (C) Diagrams depicting the primary structures 844 

of p150Glued and HPS6. Domains and binding sites in p150Glued are indicated. For 845 

HPS6 no known domains and binding sites have been identified. (D)Yeast two-hybrid 846 

assay showing the interaction of HPS6 with p150Glued C-terminus. Interaction was 847 

detected by growth of co-transformed yeast cells on high-stringency medium and the 848 

-Galactosidase colorimetric assay as previously described (Hong et al., 2009). Yeast 849 

cells co-transformed with pGBKT7 and pTD1-1, and pGBKT7-53 and pTD1-1 serve 850 

as negative and positive controls, respectively. (E) Co-IP from HEK293 cells 851 

overexpressing myc-tagged HPS6 and various Flag-tagged p150Glued deletion mutants. 852 

(F) Quantification of protein-protein interaction between HPS6 and p150Glued 853 

fragments in E was determined with NIH ImageJ. Data were from three independent 854 

co-IP experiments. (G) Yeast two-hybrid assay showing the interaction between HPS6 855 

deletion mutants and p150Glued C-terminus. (H) Co-IP from HEK293 cells 856 

overexpressing myc-tagged p150Glued and various Flag-tagged HPS6 deletion mutants. 857 

858 

Fig. 2. The perinuclear distribution of HPS6 requires an intact microtubule 859 

network and dynein-dynactin activity. (A) HeLa cells were treated with DMSO or 860 

20 M nocodazole for 1 h, fixed and immunostained with antibodies against HPS6 861 

and -tubulin. (B) HeLa cells were transiently transfected with Flag-tagged p50, 862 

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p150Glued-N (aa1-910) or p150Glued -C (aa911-1281) for 24 h and immunostained with 863 

antibodies against HPS6 and Flag. Scale bar, 10 m. 864 

865 

Fig. 3. HPS6 associates with the lysosomal compartment. (A) HeLa cells were 866 

fixed and immunostained for HPS6 and LAMP1, LAMP2, CD63, EEA1, or p230. 867 

Scale bar, 10 m. (B) Quantification of HPS6 co-localization with various markers in 868 

A. Asterisks indicate statistical significance of differences (compared with LAMP1) 869 

obtained from one-way analysis of variance with a Tukey post test. Data represent 870 

mean ± s.e.m. (n = 35-45 cells, *P < 0.05, ***P <0.001). n.s., not significant. (C-D) 871 

HPS6- or LAMP1-associated organelles were immunnoisolated from membrane 872 

fractions of HeLa cells with magnetic Dynabeads protein G conjugated with either 873 

anti-HPS6 (C) or anti-LAMP1 (D) antibody. Normal IgG and anti-GM130 serve as 874 

controls. Input and bound proteins were resolved by SDS-PAGE and detected with 875 

antibodies against EEA1, p150Glued, GM130, LAMP1, LAMP2 and HPS6. (E) 876 

Representative electron micrographs of ultrathin sections of Dynabeads from 877 

immunoisolation in C. Right panels are higher magnification images of boxed regions 878 

in left panels. Arrows and arrowheads indicate membranous structures associated with 879 

magnetic beads. Scale bars, 500 nm (left), 200 nm (right). 880 

881 

Fig. 4. The perinuclear distribution of lysosomes requires HPS6-mediated 882 

dynein-dynactin activity. (A) HeLa cells were transfected with a control 883 

non-targeting siRNA, or siRNAs against HPS6 and stained with phalloidin and 884 

antibodies against LAMP1 or LAMP2, respectively. KD, knockdown. (B) Radial 885 

profile plots of signal distribution of LAMP1 and LAMP2 relative to the nucleus in A. 886 

Shown is the distribution of fluorescent signals starting from the centroid and 887 

extending toward the cell periphery. Values are averages corresponding to the fraction 888 

of total fluorescence present in each concentric circle drawn from the centroid (Mean 889 

± s.e.m., n = 3 experiments, 20-22 cells/experiment). (C) HEK293 cells 890 

overexpressing Flag-tagged HPS5, myc-tagged HPS6, HPS6-N (aa1-700), or the 891 

internal deletion mutant of HPS6 (187-189) were lysed for co-IP with immobilized 892 

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Flag antibody. Input and bound proteins were analyzed by SDS-PAGE and 893 

immunoblotting with antibodies against Myc and Flag. (D) HPS6-depleted cells were 894 

transfected with Flag vector, Flag-tagged RNAi-resistant murine HPS6 or HPS6-N 895 

and stained with antibodies against LAMP1. (E) Radial profile plots of signal 896 

distribution of LAMP1 relative to the nucleus in D (mean ± s.e.m., n = 3 experiments, 897 

20-22 cells/experiment). (F) HeLa cells were transiently transfected with Flag-tagged 898 

HPS6 for 24 h and immunostained with antibodies against Flag and LAMP1. (G) 899 

HeLa cells were transiently transfected with Flag vector, Flag-tagged p50, p150Glued-N 900 

or p150Glued-C for 24 h and immunostained with antibodies against Flag and LAMP1. 901 

(H) Radial profile plots of signal distribution of LAMP1 relative to the nucleus in F 902 

and G (Mean ± s.e.m., n = 3 experiments, 20-22 cells/experiment). (I) 903 

Immunoisolation of LAMP1-associated membranous organelles from HeLa cells. 904 

Input and bound proteins were analyzed by SDS-PAGE and immunoblotting with 905 

antibodies to p150Glued, EEA1, LAMP1, LAMP2, HPS6, DIC, and -actin. Relative 906 

amount of p150Glued and DIC co-immunoisolated with LAMP1 was determined with 907 

NIH ImageJ. Data represent mean ± s.e.m (n = 3 experiments, **P < 0.01). Scale bars, 908 

10 m. 909 

910 

Fig. 5. Overexpression of HPS6 rescues lysosome dispersal phenotype in MEF 911 

cells from ru mouse. (A) HEK293 cells overexpressing Flag-tagged p150Glued or 912 

Flag-tagged HPS5 and myc-tagged HPS6 or myc-tagged HPS6 deletion mutant 913 

(187-189) were lysed for co-IP with immobilized Flag antibody. Input and bound 914 

proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to Flag 915 

and Myc. (B) Primary cultured MEF from WT or ru mouse were immunostained with 916 

antibodies against LAMP1 and LAMP2. (C) Immunoisolation of LAMP1-associated 917 

membranous organelles from WT or ru mouse brain. Input and bound proteins were 918 

analyzed by SDS-PAGE and immunoblotting with antibodies to p150Glued, DIC, 919 

LAMP1 and HPS6. Relative amount of p150Glued and DIC co-immunoisolated with 920 

LAMP1 was determined with NIH ImageJ. Data represent mean ± s.e.m. (n = 3 921 

experiments, *P < 0.05, **P < 0.01). (D) MEF cells from ru mouse were transfected 922 

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with constructs expressing EGFP or EGFP-HPS6 for 24 h and immunostained with 923 

antibodies against LAMP1.Scale bar, 10 m. 924 

925 

Fig. 6. Lysosomal function is impaired in HPS6-depleted cells. (A) Snapshots of 926 

control or HPS6 knockdown cells incubated with DQ-BSA for 3 h. The appearance of 927 

fluorescent spots indicates proteolytic cleavage of DQ-BSA in lysosomes. Treatment 928 

of mock-transfected cells with the vacuolar-type H+-ATPase inhibitor Bafilomycin A1 929 

(Baf) (Yoshimori et al., 1991) abolishes DQ-BSA fluorescence and serves as negative 930 

control. (B) Quantification of mean fluorescence intensity of DQ-BSA in HPS6 KD 931 

cells at different time points relative to that in control knockdown cells at 1 h in A. 932 

Data represent mean ± s.e.m. (ctrl KD, 32 cells; HPS6 KD, 48 cells. n = 3 933 

experiments, *P < 0.05, **P < 0.01). (C) Snapshots of control or HPS6 konckdown 934 

cells incubated with MR catB substrate for 2 h. Bafilomycin treatment prevents 935 

activation of cathepsin B and abolishes MR catB substrate fluorescence. (D) 936 

Quantification of MR catB substrate mean intensity relative to that of control 937 

knockdown in C. Data represent mean ± s.e.m. (ctrl KD, 49 cells; HPS6 KD, 46 cells. 938 

n = 3 experiments, ***P < 0.001). (E) Snapshots of control or HPS6 knockdown cells 939 

expressing mCherry, mCherry fused to siRNA-resistant HPS6, or HPS6-N (aa 1-700) 940 

and incubated with DQ-BSA for 1 h. (F) Quantification of DQ-BSA mean intensity 941 

relative to control knockdown in E. Data represent mean ± s.e.m. (n = 3 experiments, 942 

11-14 cells/experiment, **P < 0.01, ***P < 0.001). (G) Snapshots of control or HPS6 943 

knockdown cells expressing EGFP, EGFP fused to siRNA-resistant HPS6, or HPS6-N 944 

(aa 1-700) and incubated with MR catB substrate for 2 h. (H) Quantification of MR 945 

catB substrate mean intensity relative to control knockdown in G. Data represent 946 

mean ± s.e.m. (n = 3 experiments, 11-13 cells/experiment, ***P < 0.001). (I) 947 

Snapshots of cells expressing EGFP, EGFP fused to p150Glued-N or p150Glued -C and 948 

incubated with MR catB substrate for 2 h. (J) Quantification of MR catB substrate 949 

mean intensity relative to EGFP in I. Data represent mean ± s.e.m. (n = 3 experiments, 950 

17-24 cells/experiment, ***P < 0.001). Scale bar, 10 m. 951 

952 

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Fig. 7. Knockdown of HPS6 results in a decrease in fusion between LE/MVB and 953 

lysosome. (A) Snapshots of control or HPS6 knockdown cells preloaded with 954 

Dextran-647 to label lysosomes, and then incubated for 3 h with Dextran-488 to allow 955 

labeling of endocytic endosomes. (B) Quantification of the puncta number and mean 956 

intensity of endo-lysosomal compartments labeled with Dextran-488 or -647 and their 957 

degree of overlap in A. Data represent mean ± s.e.m. (ctrl KD, n = 38 cells; HPS6 KD, 958 

n = 39 cells. ***P <0.001). (C) Snapshots of control or HPS6 knockdown cells 959 

transfected with mCherry-LAMP1 for 24 h, and then incubated with Dextran-488 for 960 

3 h. (D) Quantification of co-localization between Dextran-488 and mCherry-LAMP1 961 

in C. Data represent mean ± s.e.m. (n = 35 cells, *P < 0.05). (E) Representative 962 

electron micrographs of control (left) or HPS6 knockdown (middle) cells. Arrows 963 

indicate MVBs, and solid arrowheads indicate lysosomes. Right panels are 964 

representative higher magnification images of structures with a typical morphology of 965 

MVB or lysosomes. (F) Quantification of indicated organelles in E. Data represent 966 

mean ± s.e.m. (ctrl KD, n = 101 cells; HPS6 KD, n = 103 cells. *P = 0.0221, ***P < 967 

0.001). (G) Cells transfected with mCherry, mCherry-p50, mCherry-p150Glued-N or 968 

mCherry-p150Glued-C for 16 h were loaded with Dextran-647 and Dextran-488 969 

sequentially as in A, stained live with Hoechst 33342 for 30 min and imaged by 970 

confocal microscope. (H) Quantification of colocalization between Dextran-488 and 971 

Dextran -647-labeled compartments in G. Data represent mean ± s.e.m. (n = 40-60 972 

cells, ***P <0.001). Scale bar for A, C and G: 10 m. 973 

974 

Fig. 8. Lysosomes fuse with LE/MVBs and mature in the perinuclear region. (A) 975 

Radial profile plots of colocalized signals between Dextran-488 and Dextran-647 in 976 

Fig. 7A. Shown is the distribution of colocalized fluorescent signals starting from the 977 

centroid and extending toward the cell periphery. Values are averages corresponding 978 

to the fraction of total fluorescence present in each concentric circle drawn from the 979 

centroid (mean ± s.e.m., n = 3 experiments, >11 cells/experiment). (B) Radial profile 980 

plots of colocalized signals between Dextran-488 and Dextran-647 in Fig. 7G (mean 981 

± s.e.m., n = 3 experiments, 15 cells/experiment). (C) Radial profile plots of signal 982 

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distribution of MR catB substrate relative to the nucleus in Fig. 6C (mean ± s.e.m., n 983 

= 3 experiments, >11 cells/experiment). (D) Radial profile plots of signal distribution 984 

of MR catB substrate relative to the nucleus in Fig. 6I (mean ± s.e.m., n = 3 985 

experiments, 15 cells/experiment). (E) Snapshots of control or HPS6 knockdown cells 986 

expressing mCherry-LAMP1 and incubated with LysoSensor DND-189 for 30 min. 987 

(F) Quantification of LysoSensor DND-189 mean intensity (left) and co-localization 988 

(Mander’s overlap) between LysoSensor DND-189 and mCherry-LAMP1 (right) in E. 989 

Data represent mean ± s.e.m. (ctrl KD, n = 31 cells; HPS6 KD, n = 40 cells. ***P < 990 

0.001). (G) Radial profile plots of LysoSensor signal distribution relative to the 991 

nucleus in E (mean ± s.e.m., n = 3 experiments, >11 cells/experiment). (H) 992 

Immunostaining analysis of HeLa cell showing Rab7 silencing with a mixture of 993 

Rab7-targeting siRNA duplexes. (I) Snapshots of control or Rab7 knockdown cells 994 

incubated with MR catB substrate for 2 h. (J) Quantification of mean intensity of MR 995 

catB substrate per cell. Data represent mean ± s.e.m. (n = 3 experiments, ***P < 996 

0.001). (K) Model for lysosomal degradation defects in the absence of HPS6. 997 

Lysosomes move along microtubules in a bidirectional manner. HPS6 serves as 998 

dynein–dynactin cargo adaptor by interacting with p150Glued and tethering the 999 

dynein–dyanctin complex to lysosomes. Depletion of HPS6 impairs retrograde 1000 

transport of lysosomes, thus resulting in cytoplasmic dispersal of lysosomes. 1001 

Moreover, it delays lysosomal degradation of endocytosed proteins, possibly resulted 1002 

from reduced fusion of LE/MVBs with perinuclear lysosomes. Scale bars, 10 m. 1003 

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