© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd doi:10.1111/tra.12249

Mucolipidosis Type IV Protein TRPML1-Dependent Lysosome Formation Austin Miller, Jessica Schafer, Cameron Upchurch, Ellen Spooner, Julie Huynh, Sebastian Hernandez, Brooke McLaughlin, Liam Oden and Hanna Fares∗ Department of Molecular and Cellular Biology, Life Sciences South Room 531, University of Arizona, Tucson, AZ 85721, USA ∗

Corresponding author: Hanna Fares, [email protected]

Abstract Lysosomes are dynamic organelles that undergo cycles of fusion and

the multiple steps of lysosome formation and identify the first regulator

fission with themselves and with other organelles. Following fusion

of this process.

with late endosomes to form hybrid organelles, lysosomes are reformed

Keywords lysosome, mucolipidosis type IV, TRPML1

as discrete organelles. This lysosome reformation or formation is a

Received 25 June 2014, revised and accepted for publication 3 Decem-

poorly understood process that has not been systematically analyzed

ber 2014, uncorrected manuscript published online 10 December 2014,

and that lacks known regulators. In this study, we quantitatively define

published online 25 January 2015

Lysosomes are membrane-bound organelles that are the major degradative compartments for endocytic, phagocytic, and autophagic materials in eukaryotic cells. This degradation is critical to many physiological processes, including processing of nutrients, down-regulation of signaling receptors, antigen presentation, killing of pathogenic organisms, and protein quality control (1–3). Lysosomes also mediate some cell death and wound repair pathways (4–8). Indeed, lysosomal dysfunction has been linked to the progression of many diseases, including a group of ˜50 known as lysosomal storage disorders (9,10).

nascent lysosomes “budding” from hybrid organelles (11). Here, we uncover a multistep pathway of lysosome formation and identify TRPML1, a non-selective cation channel protein genetically linked to the lysosomal storage disorder Mucolipidosis type IV, as the crucial regulator of the final step in the lysosome formation pathway (13).

Despite this central importance of lysosomes, the process by which lysosomes form as distinct organelles is currently unknown due to both a lack of experimental assays and known regulators. These limitations inhibit our ability to mechanistically dissect diseases associated with lysosome formation and therefore to intervene therapeutically. Lysosomes are reformed, following fusion with late endosomes to form hybrid organelles, as discrete structures by a poorly understood process that requires intra-organellar Ca2+ release (11,12). A single reported time-lapse series in NRK cells showed that lysosome reformation, which we refer to here as lysosome formation, occurs through 284 www.traffic.dk

Results Multistep lysosome formation in live RAW264.7 macrophages To visually dissect the steps of lysosome formation, we established a novel assay based on murine RAW264.7 macrophages that were loaded with BSA-AlexaFluor 594 to label late endosomes, hybrid organelles and lysosomes (because of fusion between lysosomes and late endosomes). Using spinning disk confocal microscopy to obtain time-lapse images every 1–3 seconds (Figure 1A), we identified lysosome formation events as those in which a smaller compartment (nascent lysosome) buds from a larger parent compartment (late endosome/hybrid organelle). At the higher resolution of our assay, new events in lysosome formation were uncovered. Strikingly, as the smaller compartment moves away from the

TRPML1-Dependent Lysosome Formation

parent compartment, the former remains attached to the latter by a membrane bridge, and this membrane bridge ultimately undergoes scission to release the nascent lysosome (Figure 1B,C; Movie S1, Supporting Information). The average distance traveled before scission of the nascent lysosome from the parent compartment was 0.38 ± 0.14 μm and the average time to scission was 11.2 ± 5.3 seconds. Following membrane scission, we observed some nascent lysosomes fusing with other BSA-AlexaFluor 594-labeled terminal compartments indicating that they possess the molecular machinery required for fusion (Figure 1D); our assay cannot determine whether nascent lysosomes are competent to fuse with late endosomes/hybrid organelles, other nascent lysosomes or mature lysosomes.

distance between nascent lysosomes and parent compartments was 0.57 ± 0.14 μm (Figure 2A,B; Movie S3). In the other 8/23 (35%) events, nascent lysosomes moved predominantly away from parent compartments before scission of the connecting membrane and release of the nascent lysosomes from parent compartments (Figure 2C,D; Movie S4). The average distance traveled before scission of nascent lysosomes from parent compartments was 0.63 ± 0.18 μm and the average time to scission was 43.1 ± 21.5 seconds: both the distance traveled by nascent lysosomes before scission (p 0.0009) and the time to scission (p 4.4 × 10−11 ) is significantly increased in the TRPML1-depleted than in the wild type RAW264.7 cells. This delayed scission is likely due to some TRPML1 activity remaining in cells expressing the MCOLN1 shRNA.

To determine if genes associated with lysosomal storage disorders perturb this multistep pathway of lysosome formation, we turned to TRPML1, the protein encoded by the MCOLN1 gene that has been implicated in Mucolipidosis type IV (14). We modified our assay to include GFP-TRPML1 that localizes to endo-lysosomal compartments (Figure 1E,F; Movie S2) (15). Expression of the wild type fusion protein confirmed the presence of TRPML1 at sites of lysosome formation. The average distance traveled before scission of the nascent lysosome from the parent compartment was 0.53 ± 0.24 μm and the average time to scission was 17.7 ± 9.1 seconds, which is comparable though slightly elevated to the results in RAW264.7 cells likely due to the overexpression of TRPML1.

These results suggest that TRPML1 regulates the scission step of lysosome formation. However, using MCOLN1 shRNA lines has the caveats that RNAi may affect the expression of other genes in addition to MCOLN1 and that some MCOLN1 shRNA cells have residual TRPML1 activity. We therefore immortalized mouse cells that are homozygous for a deletion in MCOLN1, thus allowing us to more accurately determine the functions of TRPML1 and its channel activity in lysosome formation.

TRPML1 is required for scission of nascent lysosomes in RAW264.7 macrophages To determine if TRPML1 influenced specific steps of lysosome formation, we imaged a RAW264.7 cell line that expresses an MCOLN1 shRNA; these cells accumulate, on the average, TRPML1 to 20% of the level found in RAW264.7 cells (15). Upon TRPML1 depletion, budding and initial movement away from parent compartments was indistinguishable from WT in all 23 recorded events (Figure 2; Movies S3 and S4). However, 15/23 (65%) events showed no scission of the connecting membrane bridge and there was bi-directional movement of nascent lysosomes away from and toward parent compartments over an average of 91.4 ± 31.2 seconds per event; the maximum Traffic 2015; 16: 284–297

Defective lysosome formation in MCOLN1 knockout cells We selected stable clones from single cells of immortalized bone marrow-derived cells from a mouse model of Mucolipidosis type IV that has a deletion in the MCOLN1 gene (Figure S1A) (16). We then made stable clones from single cells after transfection of one of the MCOLN1−/− clones (LS30) with control vector (pMIH) or a plasmid encoding GFP-TRPML1, GFP-TRPML1(D471K, D472K) or GFP-TRPML1(F465L); TRPML1(D471K, D472K) has no channel activity while TRPML1(F565L) channel activity is five to seven times lower than wild type TRPML1 (17–20). We selected clones that showed similar expression of the GFP-tagged TRPML1 variants (Figure S1B,C). In all of these clones, TRPML1 localizes predominantly to late endosomes/lysosomes, as evidenced by the limited colocalization with the early endosome marker Rab5 and the high degree of colocalization with the late endosome/lysosome marker Lamp1 (Figure S2A,B,D,E). 285

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Figure 1: Analysis of lysosome formation in RAW264.7 macrophages. A) Schematic of method used to identify lysosomal formation events. Green represents GFP-TRPML1 and red represents BSA-AlexaFluor 594 or dextran-rhodamine. ‘d’ is the distance from the edge of the parent compartment (late endosome/hybrid organelle) to the edge of the nascent lysosome. B) Cropped time-lapse images of RAW264.7 macrophages loaded with BSA-AlexaFluor 594 (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. C) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 16 independent events. Diamonds indicate scission. D) Cropped time-lapse images of RAW264.7 macrophages loaded with BSA-AlexaFluor 594 (red) at late stages of nascent lysosome formation. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, the asterisk indicates scission of the bridge, the large yellow arrow indicates a second compartment in the process of fusing with the nascent lysosomes, and the yellow asterisk indicates fusion. E) Cropped time-lapse images of RAW264.7 macrophages expressing wild type GFP-TRPML1 (green) and loaded with BSA-AlexaFluor 594 (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. F) Quantification of the movement of nascent lysosomes relative to parent compartments over time for ten independent events. 286

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TRPML1-Dependent Lysosome Formation

Figure 2: Analysis of lysosome formation in RAW264.7 macrophages expressing MCOLN1 shRNA. A) Cropped time-lapse images of MCOLN1 RNAi cells loaded with BSA-AlexaFluor 594. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. B) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 15 independent events. C) Cropped time-lapse images of MCOLN1 RNAi cells loaded with BSA-AlexaFluor 594. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. D) Quantification of the movement of nascent lysosomes relative to parent compartments over time for eight independent events. Traffic 2015; 16: 284–297

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Figure 3: Lysosome formation in MCOLN1−/− cells expressing GFP-TRPML1 or in MCOLN1−/− cells. A) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1 (green) and pre-loaded with dextran-rhodamine (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. B) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 15 independent events. Diamonds indicate scission. C) Cropped time-lapse images of MCOLN1−/− + pMIH cells pre-loaded with dextran-rhodamine (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. D) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 15 independent events. 288

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TRPML1-Dependent Lysosome Formation

Using live imaging of the MCOLN1−/− cells expressing wild type GFP-TRPML1 and pre-loaded with dextran-rhodamine, we detected the same multistep pathway of lysosome formation: nascent lysosomes budding off parent compartments, moving away from the latter while being connected by a bridge, and scission of the connecting bridge in 15 of 15 recorded events (Figure 3A, Movie S5). The average distance traveled before scission of the nascent lysosome from the parent compartment was 0.45 ± 0.26 μm and the average time to scission was 16.8 ± 4.7 seconds (Figures 3B and 5C,D). This distance and time to scission are similar to what we observed in RAW264.7 macrophages (Figure 1B). The parent compartments and the nascent lysosomes in these cells stain with LysoTracker Red and are therefore acidic (Figure S3A).

dextran-rhodamine. These TRPML1 mutant cells were similar to MCOLN1−/− cells; there was normal nascent lysosome formation and initial movement away from parent compartments, indicating that TRPML1 channel activity is not required for these events (Figure 4A; Movie S7). In addition, we could not detect scission of the membrane connecting nascent lysosomes to parent compartments in 18 of 18 events over the recorded time, which was on the average 156.8 ± 54.9 seconds and the nascent lysosomes showed back-and-forth movement, indicating a requirement of TRPML1 channel activity for directed movement of nascent lysosomes and for scission of the connecting membrane (Figures 4B and 5D; Movie S7). Over this time, the maximum distance between nascent lysosomes and parent compartments was 0.5 ± 0.3 μm (Figure 5C).

MCOLN1−/− cells carrying the control vector pMIH and pre-loaded with dextran-rhodamine showed normal nascent lysosome formation and initial movement away from parent compartments, indicating that TRPML1 is not required for these steps of lysosome formation (Figure 3C). However, we could not detect scission of the membrane connecting nascent lysosomes to parent compartments in 15 out of 15 events over the recorded time, which was on the average 68.3 ± 11.1 seconds (Figures 3D and 5D; Movie S6). Furthermore, after initially moving away from the parent compartments, the nascent lysosomes moved back toward the parent compartments, which is in contrast to cells expressing wild type TRPML1 where nascent lysosomes showed directed movement away from parent compartments (Figure 3B,D); this suggests that TRPML1 is required to orchestrate directed movement of nascent lysosomes. Over this time, the maximum distance between nascent lysosomes and parent compartments was 0.6 ± 0.2 μm in MCOLN1−/− cells (Figure 5C). Similar to MCOLN1−/− cells expressing GFP-TRPML1, the parent compartments and the nascent lysosomes in MCOLN1−/− cells also stain with LysoTracker Red and are therefore acidic (Figure S3B).

To probe the Ca2+ requirement for lysosome formation in cells that had TRPML1 activity, we exposed MCOLN1−/− cells expressing wild type GFP-TRPML1 and pre-loaded with dextran-rhodamine to DMSO (control) or to the cell-permeant chelator BAPTA-AM. DMSO had no effect on lysosome formation yielding similar results to cells without DMSO (Figure S4A): the average distance traveled before scission of the nascent lysosome from the parent compartment was 0.43 ± 0.06 μm and the average time to scission was 16.6 ± 8.1 seconds (Figure S4B). In contrast, BAPTA-AM gave similar lysosome formation defects as MCOLN1−/− cells expressing the channel-dead TRPML1(D471K, D472K) (Figure S4C): we could not detect scission of the membrane connecting nascent lysosomes to parent compartments in 12 of 12 events over the recorded time, which was on the average 105.2 ± 24.79 seconds (Figure S4D). Thus, blocking Ca2+ activity in wild type cells that did not have the time to accumulate significant lysosomal defects after the relatively short exposure to BAPTA-AM also causes defects in lysosome formation. This suggests that TRPML1 functions directly in lysosome formation and that the lysosome formation defects in cells lacking TRPML1 is not due to defects in the parent compartments (see below) from which nascent lysosomes bud.

TRPML1 channel functions during lysosome formation TRPML1 is a channel protein. To determine if this activity is required for lysosome formation, we created MCOLN1−/− cells expressing the channel-dead GFP-TRPML1(D471K, D472K) and pre-loaded them with Traffic 2015; 16: 284–297

To examine this dependence of the late stages of lysosome formation on TRPML1 channel activity using another mutant, we created MCOLN1−/− cells expressing 289

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Figure 4: Lysosome formation in MCOLN1−/− cells expressing GFP-TRPML1 channel mutants. A) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1(D471K, D472K) (green) and pre-loaded with dextran-rhodamine (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. B) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 18 independent events. C) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1(F465L) (green) and pre-loaded with dextran-rhodamine (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. D) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 13 independent events. 290

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TRPML1-Dependent Lysosome Formation

Figure 5: Analysis of lysosome formation. A) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1(F465L) (green) and pre-loaded with dextran-rhodamine (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. B) Quantification of the movement of nascent lysosomes relative to parent compartments over time for eleven independent events. Diamonds indicate scission. C–G) Averages and standard deviations for maximum distance between nascent lysosomes and parent compartments before scission or for the duration of the imaging when there is no scission (C), time to scission or duration of imaging from the appearance of a nascent lysosome when there is no scission (D), sizes of nascent lysosomes (E), sizes of parent compartments (F), and initial velocities of nascent lysosomes moving away from parent compartments (G). Red arrows indicate that quantitation was performed when there was scission of the connecting membrane, which is 15/15 (100%) of lysosome formation events in wild type TRPML1-expressing and 11/24 (45.8%) in TRPML1(F465L)-expressing cells. Inverted brackets indicate Student t -test probabilities that are less than 0.05. Traffic 2015; 16: 284–297

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GFP-TRPML1(F465L), which has reduced channel activity and is one of the mutations causing Mucolipidosis type IV. These cells were pre-loaded with dextran-rhodamine, and live-cell imaging showed an intermediate phenotype that was similar to RAW264.7 macrophages with reduced MCOLN1 levels. There was normal lysosome formation and initial movement away from parent compartments in all 24 recorded events (Figures 4C,D and 5A,B; Movies S8 and S9). However, 13 of 24 (54.2%) events showed a similar phenotype to MCOLN1−/− cells such that there was no scission of the connecting membrane and bi-directional movement of nascent lysosomes over an average of 135.6 ± 44.1 seconds per event; the maximum distance between nascent lysosomes and parent compartments was 0.6 ± 0.5 μm (Figure 4C,D; Movie S8). However, in 11 of 24 (45.8%) events, there was unidirectional movement and scission of the connecting membrane releasing nascent lysosomes from parent compartments (Figure 5A,B; Movie S9). The average distance traveled before scission of nascent lysosomes from parent compartments was 0.4 ± 0.08 μm and the average time to scission was 45.1 ± 17.8 seconds: while the distance traveled by nascent lysosomes before scission is similar to cells expressing wild type TRPML1 (p 0.85), the time to scission is significantly higher (p 9.2 × 10−5 ) than in cells expressing wild type TRPML1 (Figures 5C and S5). This delay in scission suggests that TRPML1 channel activity directly regulates mechanisms that mediate scission of nascent lysosomes from parent compartments. Parameters of lysosome formation in wild type and mutant cells To rule out the possibility that TRPML1 channel activity simply mediates lysosome formation by impacting the size of nascent lysosomes, we compared compartment sizes in our images. The different MCOLN1−/− cells expressing pMIH or TRPML1 variants, with the exception of TRPML1(F465L) which show slightly increased sizes relative to wild type TRPML1-expressing cells, were all similar (Figures 5E and S5). Thus, these results indicate that TRPML1 protein is not required for establishing the sizes of nascent lysosomes budding from parent compartments. Nonetheless, the defect in the scission of membranes connecting nascent lysosomes to parent compartments should result in an increase in the sizes of the parent compartments 292

(hybrid organelles). Indeed, there was a statistically significant increase in the sizes of parent compartments in MCOLN1−/− cells carrying pMIH relative to MCOLN1−/− cells expressing wild type TRPML1 or TRPML1(D471K, D472K) (Figures 5F and S5). MCOLN1−/− cells expressing TRPML1(F465L) showed an intermediate phenotype such that parent compartment sizes were statistically similar to cells carrying pMIH or expressing wild type TRPML1 (Figures 5F and S5). This increase in parent compartment sizes in MCOLN1−/− cells carrying pMIH is consistent with a defect in lysosome formation and thus represents the first abnormality along the endocytic pathway: there was no difference in early endosomal sizes between all the cell lines using an RFP-Rab5 marker, which is consistent with a previous study that showed normal early endosomal size and function in Mucolipidosis type IV cells (Figure S2A,C) (21). Furthermore, the lack of increase in parent compartment size in cells expressing the channel-defective TRPML1(D471K, D472K) suggest that the connecting membrane eventually ruptures in these cells (possibly due to mechanical shear or compensating activities by the homologous TRPML2 and/or TRPML3 proteins). This difference in parent compartment sizes between MCOLN1−/− cells carrying pMIH relative to MCOLN1−/− cells expressing TRPML1(D471K, D472K) suggests that TRPML1 protein has functions in lysosome formation that go beyond its channel activity, e.g. by recruiting other proteins to this process. This channel-independent function of TRPML1 is further highlighted by the increased initial velocity of movement of nascent lysosomes away from parent compartments in MCOLN1−/− cells carrying pMIH relative to any of the TRPML1 variants (Figures 5G and S5). Furthermore, this increased velocity suggests additional functions for TRPML1 in coordinating the movement of nascent lysosomes away from parent compartments.

Discussion A system for analyzing lysosome formation We have established a system to measure parameters of lysosome formation that include ‘budding’ of a nascent lysosome from a parent compartment (late endosome/hybrid organelle), directed movement of the nascent lysosome away from the parent compartment through the elongation or extension of a membrane Traffic 2015; 16: 284–297

TRPML1-Dependent Lysosome Formation

bridge, and scission of the bridge to release a distinct lysosome; we refer to this structure as a nascent lysosome because it has a diameter of 0.3–0.4 μm, smaller than the 1–2 μm diameters of mature lysosomes. We think that the fusion of nascent lysosomes with each other and/or with existing lysosomes results in primary and secondary lysosomes. This process we are describing is likely the same one used to generate discrete lysosomes after endocytic delivery of material from the plasma membrane to late endosomes (lysosome formation), after delivery of biosynthetic material from the Golgi apparatus to late endosomes (lysosome biogenesis), after fusion of autophagosomes with late endosomes (autophagic lysosome reformation), or after fusion of lysosomes with late endosomes (lysosome reformation) (3,11,15,22).

structure is actin. Indeed, lysosome formation is quite similar topologically to endocytic vesicle formation at the plasma membrane where actin polymerization is required (24). There are preliminary data that suggest actin involvement in lysosome formation. Loss of the small GTPase RAB-2/UNC-108 results in a similar lysosome formation defect as loss of CUP-5 in C. elegans (25); mammalian Rac2, an actin regulator and RAB-2 homologue, physically associates with TRPML1 (26). Furthermore, another potential actin regulator, phosphatidylinositol-4,5-bisphosphate was identified as essential for autophagic lysosome reformation (22). Indeed, phosphatidylinositol-4-phosphate 5-kinases A and B were shown to be required for autophagic lysosome reformation of which at least one that was tested physically associates with TRPML1 (22,26).

TRPML1 requirement for lysosome formation We have also identified TRPML1 as the first mammalian regulator of this lysosome formation mediating activities that are required for the directed movement of nascent lysosomes and the scission of the membrane bridge. These results are consistent with our previous studies in Caenorhabditis elegans showing that CUP-5, the ortholog of mammalian TRPML1, is required for the transport of endocytosed solutes to lysosomes (23).

Lysosome formation and Mucolipidosis type IV Lysosome formation is the first defect in the endocytic pathway that has been detected so far in cells lacking TRPML1. However, Mucolipidosis type IV cells show additional defects, including iron and zinc homeostasis, lysosome exocytosis, lipid transport from the late endosomes to the Golgi apparatus, swollen lysosomes, lysosomal transport and degradation and autophagy (16,17,21,27–35). Based on the data after exposure to BAPTA-AM and in cells expressing TRPML1 channel mutant, this defective lysosome formation likely occurs early in the cellular pathology and leads to some of the other defects in Mucolipidosis type IV cells. Further studies will be needed to elaborate the cascade of events that lead from loss of TRPML1 to the cellular abnormalities and symptoms in Mucolipidosis type IV patients.

We think that TRPML1 has a direct function in lysosome formation because the defect in lysosome formation in cells lacking TRPML1 is the first perturbation observed in the endocytic pathway in these cells. Indeed, in C. elegans lacking CUP-5, there is a similar block in nascent lysosomes being released from normal-sized parent compartments, suggesting that defects in the parent compartments are not leading to defects in lysosome formation (23). Similarly, we see a defect in lysosome formation in MCOLN1−/− cells expressing TRPML1 channel mutants and that have parent compartments with normal sizes. TRPML1 is a good candidate for the channel that releases the intra-organellar Ca2+ that is required for lysosome formation, as shown in our live study and as previously shown in a cell-free assay (12). This Ca2+ likely regulates proteins required for the extension and/or scission of the membrane bridge connecting parent compartments to nascent lysosomes. A good candidate for a Ca2+ -regulated Traffic 2015; 16: 284–297

Materials and Methods Cell culture and transfection Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 2 mM Glutamax and supplemented with 10% Fetal Bovine Serum, 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen) at 37∘ C in 95% air at 5% carbon dioxide. Stable clones LS5 (expressing GFP-TRPML1) and LS9 (expressing MCOLN1 shRNA) of mouse RAW264.7 macrophages (ATCC) included G418 at 250 μg/mL in the medium (15). Stable clones of the mouse MCOLN1−/− immortalized cells with re-introduced plasmids included hygromycin (Invitrogen) at 100 μg/mL in the medium. Transfections were performed using Fugene HD (Roche).

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Immortalizing MCOLN1−/− cells and making stable clones

were incubated in 1 μM LysoTracker Red DND-99 (Life Technologies) for

Bone marrow isolated from legs of MCOLN1−/− mice were grown in the presence of 10 ng/mL murine GM-CSF (Peprotech) for 3 days and infected with a pLenti-SV40 lentivirus that expresses the SV40 large T-antigen (ABM; 16). After 2 months, living cells were grown in the absence of GM-CSF. Clones originating from single cells that grew in the absence of GM-CSF were isolated by dilution in 96-well plates and frozen after confirming by PCR that they were homozygous for the MCOLN1 deletion; one clone called LS30 was selected for further study.

4 h in the tissue culture incubator. Cells were then washed twice with pre-heated medium lacking Phenol Red and left in 2 mL of the same medium for live imaging. LS49 (MCOLN1−/− + pMIH) cells were first pre-labeled with dextran-FITC MW 10 000 (Sigma-Aldrich) as described above for dextran-rhodamine before the LysoTracker staining and imaging. For the BAPTA-AM inhibitor studies, LS44 (MCOLN1−/− + GFPTRPML1) cells that were pre-loaded with dextran-rhodamine were exposed to 33 μM BAPTA-AM (dissolved in DMSO; Sigma-Aldrich) at 37∘ C for 30 min prior to imaging. Control cells received the same treatment using the same amount of DMSO.

Introducing GFP-TRPML1 into immortalized MCOLN1−/− cells A

stable

MCOLN1−/−

clone

(LS30)

was

transfected

with

linearized plasmid DNA, either the control vector pMSCV.IREShyg (pMIH), or GFP-TRPML1 wild type or channel mutants cloned into this vector (36). Selection was carried out in medium containing hygromycin at 200 μg/mL until the control plate (no DNA) showed 100% lethality. Stable clones from single cells were then selected and maintained in medium containing 100 μg/mL hygromycin. Confocal imaging was then performed on individual clones using the same microscopy conditions to compare levels of GFP-TRPML1 in the cells; the GFP intensity of 50–60 GFP-TRPML1 positive structures from each strain were analyzed using METAMORPH software and cells that showed comparable levels of GFP-TRPML1 were used in subsequent studies. These cell lines are:

LS49: LS30 + pMIH LS44: LS30 + pHD756 [GFP-TRPML1] LS39: LS30 + pHD838 [GFP-TRPML1(D471K, D472K)] LS52: LS30 + pHD857 [GFP-TRPML1(F465L)]

For RAW264.7 cell lines, a similar procedure was used except that 7.5 × 104 cells in 90 μL of medium lacking Phenol Red were mixed with 10 μL of 10 mg/mL BSA-AlexaFluor 94 (Life Technologies) dissolved in 1× PBS in the center of a 35 mm glass bottom culture dish with a No. 0 coverglass (MatTek Corporation). After 30 min in the tissue culture incubator, 2 mL of medium lacking Phenol Red were added and the cells were imaged and analyzed.

RFP-Rab5 imaging Cells were transfected using Turbofect (Life Technologies) with a plasmid expressing RFP-Rab5 (Origene). Live cells expressing the lowest detectable levels of RFP-Rab5 were then imaged to determine sizes of Rab5 compartments and to determine the percent colocalization between TRPML1 and Rab5 in each cell line, which is the number of structures that contained both GFP-TRPML1 and RFP-Rab5 divided by the total number of structures that contain GFP-TRPML1.

Lamp1 immunofluorescence Cells were fixed and processed as previously described (15). Primary

Live imaging assay For MCOLN1−/− cell lines dextran-rhodamine imaging, 3 × 105 cells in 90 μL of medium were mixed with 10 μL of 10 mg/mL dextran-rhodamine, MW 10 000 (Sigma-Aldrich) dissolved in 1× PBS in the center of a 35 mm glass bottom culture dish with a No. 0 coverglass (MatTek Corporation). After 1 h in the tissue culture incubator, 2 mL of medium were added and the cells were left overnight. Before imaging, cells were washed twice with pre-heated medium lacking Phenol Red and then left in 2 mL of the same medium. Imaging was carried out at room temperature and images from three z-planes that were 0.6 μm apart were acquired every ∼3 seconds for 1–2 min. SlideBook 5.5 (Intelligent Imaging Innovations) was used to generate a sum intensity projection of images from these z-planes; these were analyzed using SLIDEBOOK 5.0 software (Intelligent Imaging Innovations). A similar procedure was used for staining MCOLN1−/− cell lines with LysoTracker for live imaging. LS44 (MCOLN1−/− + GFP-TRPML1) cells

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antibodies used were chicken anti-GFP (Abcam) and rabbit anti-Lamp1 (Abcam), and secondary antibodies were donkey anti-chicken-FITC and goat anti-rabbit-Cy3 (Abcam). Three z-planes from seven cells from each strain were imaged by confocal microscopy and the sum intensity projection from each stack was used to determine percent colocalization in each cell of GFP-TRPML1 with Lamp1 expressed as the number of structures that contained both GFP-TRPML1 and Lamp1 divided by the total number of structures that contain GFP-TRPML1.

Microscopy Confocal images were collected using an Intelligent Imaging Innovations (3i) System built on a Marianas (Zeiss, Germany) microscope base with a Z-piezo stage (ASI PZ2150FT), Yokogawa CSU-X1M Spinning Disk, 488 nm laser, 561 nm laser, 100× Plan APO Objective and a Photometrics Evolve 512 CCD. RFP-Rab5 confocal images were taken on a Zeiss LSM510 (Carl Zeiss) laser scanning confocal, using software.

LSM

imaging

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Molecular methods Standard methods were used for the manipulation of recombinant DNA (37). PCR was performed using the Expand Long Template PCR System (Roche) according to the manufacturer’s instructions. Sire-directed mutagenesis was performed using QuikChange XL (Agilent Technologies). All other enzymes were from New England Biolabs.

of scission of membranes connecting parent compartments to nascent lysosomes. Movie S3: RAW264.7 MCOLN1 shRNA + BSA-Alexa 594 (red) − no scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes.

Plasmids Inserts of all plasmids were sequenced to confirm that only the desired changes were introduced. The following plasmids were made and used in this study:

Movie S4: RAW264.7 MCOLN1 shRNA + BSA-Alexa 594 (red) − scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes.

pHD756: Encodes GFP fused to mouse wild type TRPML1 expressed under the control of the MSCV promoter in the pMIH vector. pHD838: Encodes GFP fused to mouse TRPML1(D471K, D472K) mutant expressed under the control of the MSCV promoter in the pMIH vector. pHD857: Encodes GFP fused to mouse TRPML1(F465L) mutant expressed under the control of the MSCV promoter in the pMIH vector.

Plasmid sequences and details on how these plasmids were constructed are available upon request.

Statistical methods Student’s t-test was used to compare measurements from two samples using a two-tailed distribution (Tails = 2) and a two-sample unequal variance (Type = 2).

Acknowledgments We thank Susan Slaugenhaupt (Massachusetts General Hospital) for dissected legs from the MCOLN1−/− mice and Brooke Beam (W.M. Keck Center for Surface and Interface Imaging in the Department of Chemistry and Biochemistry, University of Arizona) for microscopy assistance. This work was supported by a National Science Foundation grant number 1052166 to H. F. The authors declare no competing financial interests.

Supporting Information Additional Supporting Information may be found in the online version of this article: Movie S1: RAW264.7 + BSA-Alexa 594 (red) − scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Movie S2: RAW264.7 + GFP-TRPML1 (green) + BSA-Alexa 594 (red) − scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time

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Movie S5: MCOLN1−/− + wild type GFP-TRPML1 (green) + Dextran-Rhodamine (red) − scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Movie S6: MCOLN1−/− + Dextran-Rhodamine (red) − no scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Movie S7: MCOLN1−/− + GFP-TRPML1(D471K, D472K) (green) + Dextran-Rhodamine (red) − no scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Movie S8: MCOLN1−/− + GFP-TRPML1(F465L) (green) + DextranRhodamine (red) − no scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Movie S9: MCOLN1−/− + GFP-TRPML1(F465L) (green) + DextranRhodamine (red) − scission. Large red arrows indicate parent compartments, small white arrows indicate nascent lysosomes, and asterisks appear at time of scission of membranes connecting parent compartments to nascent lysosomes. Figure S1: Establishing homogenous cell lines lacking TRPML1 or expressing TRPML1 variants. A) Schematic of the procedure used to immortalize bone marrow-derived cells from MCOLN1−/− mice. The gel to the right shows the expected 400 bp band from wild type (WT) and the 187 bp MCOLN1 deletion band after PCR of genomic DNA from wild type or two independently isolated MCOLN1−/− clones (LS27 and LS30). B) Confocal images of live MCOLN1−/− cells with the pMIH control vector or expressing the indicated GFP-TRPML1 protein. Images were acquired using the same microscopy conditions. C) Quantification of the GFP intensities per unit area of cells shown in (B). Wild type GFP-TRPML1 levels were set to 1.

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Figure S2: Analysis of endosomal and lysosomal markers. A) Confocal images of MCOLN1−/− cells expressing pMIH (control plasmid-No) or the indicated GFP-TRPML1 variant (green) and RFP-Rab5 (red). B) Quantification of the percent colocalization between GFP-TRPML1 variants and RFP-Rab5 from (A). C) Quantification of the sizes of RFP-Rab5 compartments from (A). D) Confocal images of fixed cells immunostained to detect GFP-TRPML1 variants and Lamp1. E) Quantification of the percent colocalization between GFP-TRPML1 variants and Lamp1 from (D). Figure S3: Acidity of Compartments in MCOLN1−/− cells expressing GFP-TRPML1 or in MCOLN1−/− cells. A) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1 (green) and stained with Lysotracker Red (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. B) Cropped time-lapse images of MCOLN1−/− + pMIH cells pre-loaded with dextran-FITC (green) and stained with Lysotracker Red (red). Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. Figure S4: Effect of Ca2+ inhibition on lysosome formation. A) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1 (green) and pre-loaded with dextran-rhodamine (red) after exposure to DMSO. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome, and the asterisk indicates scission of the bridge. B) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 12 independent events. Diamonds indicate scission. C) Cropped time-lapse images of MCOLN1−/− cells expressing wild type GFP-TRPML1 (green) and pre-loaded with dextran-rhodamine (red) after exposure to BAPTA-AM. Arrowheads indicate parent compartments, large arrows indicate nascent lysosomes, small arrows indicate bridge connecting parent compartment to nascent lysosome. There was no scission observed over the recorded time. D) Quantification of the movement of nascent lysosomes relative to parent compartments over time for 12 independent events. Figure S5: Statistical analysis of lysosome biogenesis parameters. Comparison of nascent compartment areas, parent compartment areas and initial velocities of nascent lysosomes in MCOLN1−/− cells with the pMIH control vector (KO) or expressing the indicated GFP-TRPML1 variants. F465L* indicates events where no scission was observed.

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Mucolipidosis type IV protein TRPML1-dependent lysosome formation.

Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endos...
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