A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway

Mallika Bhat; Akshaya Nambiar; Lakshmi Edakkandiyil; Irine Maria Abraham; Ritoprova Sen; Mamta Negi; Ravi Manjithaya

doi:10.1091/mbc.E23-09-0344

. 2024 Jul 11;35(8):mr6. doi: 10.1091/mbc.E23-09-0344

A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway

Mallika Bhat ^a, Akshaya Nambiar ^a, Lakshmi Edakkandiyil ^b, Irine Maria Abraham ^a, Ritoprova Sen ^a, Mamta Negi ^a, Ravi Manjithaya ^a,^c,^*

Editor: Sergio Grinstein^d

PMCID: PMC11321044 PMID: 38888935

Abstract

Maintenance of a pool of active lysosomes with acidic pH and degradative hydrolases is crucial for cell health. Abnormalities in lysosomal function are closely linked to diseases, such as lysosomal storage disorders, neurodegeneration, intracellular infections, and cancer among others. Emerging body of research suggests the malfunction of lysosomal hydrolase trafficking pathway to be a common denominator of several disease pathologies. However, available conventional tools to assess lysosomal hydrolase trafficking are insufficient and fail to provide a comprehensive picture about the trafficking flux and location of lysosomal hydrolases. To address some of the shortcomings, we designed a genetically-encoded fluorescent reporter containing a lysosomal hydrolase tandemly tagged with pH sensitive and insensitive fluorescent proteins, which can spatiotemporally trace the trafficking of lysosomal hydrolases. As a proof of principle, we demonstrate that the reporter can detect perturbations in hydrolase trafficking, that are induced by pharmacological manipulations and pathophysiological conditions like intracellular protein aggregates. This reporter can effectively serve as a probe for mapping the mechanistic intricacies of hydrolase trafficking pathway in health and disease and is a utilitarian tool to identify genetic and pharmacological modulators of this pathway, with potential therapeutic implications.

Recent research has highlighted the relevance of mannose-6-phosphate (M6P) pathway mediated trafficking of lysosomal hydrolases in health and disease. Existing assays to monitor this pathway are inadequate in various respects.
The authors have developed a new genetically-encoded fluorescence-based reporter to spatiotemporally track M6P pathway. Functionality and robustness of the reporter was validated under different physiological and pathophysiological conditions and also across different cell types.
Simple and utilitarian design of the reporter enables its potential use in large scale screens aimed at identifying pharmacological and genetic modulators of the pathway of putative therapeutic relevance.

INTRODUCTION

Lysosome is a key organelle responsible for the degradation and recycling of cellular components and the regulation of cell metabolism (Ballabio and Bonifacino, 2020; Shin and Zoncu, 2020; Bouhamdani et al., 2021). The degradative function of lysosomes is carried out by lysosomal hydrolases, a group of more than 60 degradative enzymes, such as proteases, nucleases, lipases, glycosidases, sulfatases, phosphatases, and phospholipases. These hydrolases show high enzymatic activity at a low pH of 4.5–5.0, which is a characteristic feature of the lysosomal lumen (Coffey and De Duve, 1968; Ohkuma and Poole, 1978; Hosogi et al., 2014). The trafficking of lysosomal hydrolases to the lysosomal compartments from the endoplasmic reticulum (ER) is an important step toward generating a functional lysosome. This trafficking is mediated by receptors such as mannose-6-phosphate receptors (M6PRs), lysosomal integral membrane protein-2 (LIMP-2), sortilin, and low-density lipoprotein receptors (LDL receptors) (Storch and Braulke, 2005; Reczek et al., 2007; Braulke and Bonifacino, 2009; Markmann et al., 2015; Mitok et al., 2022). Majority of lysosomal hydrolases are transported to the lysosomes by M6PRs. This process initiates with the recognition of M6P-tagged lysosomal hydrolases by M6P receptors (M6PRs) in the trans-Golgi network (TGN) (Figure 1A). The M6PR–hydrolase complexes traverse through TGN-derived clathrin-coated vesicles and early endosomes to eventually reach late endosomes. Acidic pH in the late endosomes causes dissociation of the hydrolases from the M6PRs, and the M6PRs are transported back to the TGN to facilitate subsequent rounds of hydrolase trafficking. Late endosomes mature into or fuse with lysosomes, resulting in delivery of the hydrolases to the lysosomes. Two M6PRs: cation-dependent (CD-M6PRs) and cation-independent (CI-M6PRs) together are responsible for this process.

FIGURE 1: — Tracking M6PRs: a routinely used method to monitor lysosomal hydrolase trafficking. (A) Schematic representation of M6P pathway. (B) Representative microscopy images of HeLa cells stained with MitoTracker/transfected with subcellular organelle marker constructs (mCherry-TGNP-N-10, mRFP-Rab5, mCherry-Rab7A, Lamp1-RFP, dsRed-Rab11) and CFP-CDM6PR. Scale bar, 10 μm. (C) Representative microscopy images of HeLa cells stained with MitoTracker/transfected with subcellular organelle marker constructs (mCherry-TGNP-N-10, mRFP-Rab5, mCherry-Rab7A, Lamp1-RFP, dsRed-Rab11) and immunostained for CI-M6PR. Scale bar, 10 μm. (D) Quantitation of Mander’s coefficients between CD-M6PR and subcellular organelle markers. M1 = Fraction of CD-M6PR colocalizing with subcellular organelle marker. M2 = Fraction of subcellular organelle marker colocalizing with CD-M6PR. (n ≥ 25, N = 3). M1 for MitoTracker compared with M1 for all the organelle markers. Also, M2 for MitoTracker compared with M2 for all the organelle markers. ****, p < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test, mean ± SEM. (E) Quantitation of Mander’s coefficients between CI-M6PR and subcellular organelle markers. M1 = Fraction of CI-M6PR colocalizing with subcellular organelle marker. M2 = Fraction of subcellular organelle marker colocalizing with CI-M6PR. (n ≥ 25, N = 3). M1 for MitoTracker compared with M1 for all the organelle markers. Also, M2 for MitoTracker compared with M2 for all the organelle markers. ****, p < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test, mean ± SEM.

The multifaceted role of lysosomes in a cell warrants the maintenance of a pool of functional lysosomes to ensure cellular homeostasis. Lysosomal malfunctioning contributes to several pathologies such as lysosomal storage disorders (LSDs) (Micsenyi and Walkley, 2022), neurodegeneration (Finkbeiner, 2020; Udayar et al., 2022), intracellular infections (Sachdeva and Sundaramurthy, 2020; Bird et al., 2023), cancer (Davidson and Vander Heiden, 2017; Tang et al., 2020), cardiovascular diseases (Chi et al., 2020; Bhat and Li, 2021), and autoimmune diseases (Ge et al., 2015; Gros and Muller, 2023).

Lysosomal dysfunction in diseased state arises from a variety of factors such as alterations in the expression of lysosome-related genes, loss of acidity within the lysosomal compartment, mutations in genes encoding lysosomal hydrolases and membrane proteins, defects in the delivery of substrates for lysosomal degradation and perturbed trafficking of lysosomal hydrolases (Bonam et al., 2019; Bouhamdani et al., 2021). Studies over the past two decades have highlighted the role of perturbations in the trafficking of lysosomal hydrolases to lysosomes as a vital contributing factor in lysosomal dysfunction in several diseases. Pathogenic mutations associated with debilitating LSDs such as neuronal ceroid lipofuscinosis (Metcalf et al., 2008; Mamo et al., 2012; Yasa et al., 2020, 2021; Wang et al., 2022; Calcagni’ et al., 2023), mucolipidosis type II, III, and IV (Khan and Tomatsu, 2020; Richards et al., 2022; Zhang et al., 2022), spondyloepiphyseal skeletal dysplasia (Kondo et al., 2018; Carvalho et al., 2020), and Gaucher disease (Patel et al., 2023) cause a block in the trafficking of lysosomal hydrolases resulting in the accumulation of undigested material in the lysosomes. Perturbed hydrolase trafficking is also indicated to play a role in neurodegenerative disorders such as Parkinson’s disease (Matrone et al., 2016; Mazzulli et al., 2016; Kim et al., 2017; Waschbüsch et al., 2019; Beilina et al., 2020; Wu et al., 2020) and hereditary spastic paraplegia (Hirst et al., 2018). Retromer complex is a crucial component of the lysosomal hydrolase trafficking pathway. Loss of function of retromer complex is implicated in the formation of protein aggregates like α-synuclein, amyloid β and tau, thereby contributing to diseases such as Parkinson’s disease, Alzheimer’s disease, Pick’s disease, and progressive supranuclear palsy (Carosi et al., 2021). Apart from neurodegenerative disorders, intracellular bacterial pathogens such as Salmonella, Legionella, Chlamydia, Helicobacter, and Shigella have also been reported to interfere with the hydrolase trafficking pathway for their enhanced survival and replication in the host cell (McGourty et al., 2012; Finsel et al., 2013; Bärlocher et al., 2017; Elwell et al., 2017; Khan and Tomatsu, 2020; Sun et al., 2021). Furthermore, some viruses utilize lysosomal cathepsins for their activation and cellular entry. Thus, defective hydrolase trafficking is detrimental to their replication within host cells (Richards et al., 2022). Modulated hydrolase trafficking flux may have a role in providing nutrients to cancer cells and promoting their invasive and metastatic character (Sleat et al., 1995; Yang et al., 2011; Sevenich and Joyce, 2014; Olson and Joyce, 2015; Kovalyova et al., 2022; Pechincha et al., 2022).

Given the relevance of lysosomal hydrolase trafficking in diseases, a detailed understanding of the mechanism and regulation of this pathway in healthy and diseased states is crucial. Toward this end, a probe that can inform about the trafficking flux of lysosomal hydrolases, their localization, and their functionality is desirable. Conventionally used tools to study lysosomal hydrolase trafficking include, immunoblotting for immature and mature forms of hydrolases, fluorography of radiolabeled hydrolases, and tracking the localization of the prime receptors of hydrolase trafficking M6PRs (Scheel et al., 1990; Waguri et al., 2003; Seaman, 2004; Anitei et al., 2014; Breusegem and Seaman, 2014; Tavares and daSilva, 2017; Cuddy and Mazzulli, 2021). These tools do not provide direct information about the localization of lysosomal hydrolases in the cell. Additionally, these assays are time consuming and tedious, and hence are not suitable for large-scale screens to identify hydrolase trafficking modulators. To overcome the limitations of the existing assays, we have developed a new fluorescent reporter to track the flux of lysosomal hydrolase trafficking. This reporter enables time-resolved monitoring of the entire journey of lysosomal hydrolases from the ER to the lysosomes. In addition, it enables quantification of the relative number of pre-lysosomal and lysosomal vesicles containing hydrolases. This reporter will be useful in studying the mechanistic details of the lysosomal hydrolase trafficking pathway and can be optimized for large-scale screens to identify therapeutically relevant genetic and pharmacological regulators of the pathway.

RESULTS

Tracking M6PRs: a routinely used method to monitor lysosomal hydrolase trafficking

Because M6PRs play a predominant role in hydrolase trafficking, following their intracellular distribution is a commonly used method to detect changes as well as identify modulators of hydrolase trafficking (Hirst et al., 1998; Waguri et al., 2003; Seaman, 2004; Anitei et al., 2014; Breusegem and Seaman, 2014). Here, we have followed the hydrolase trafficking pathway by monitoring subcellular distribution of M6PRs using fluorescent microscopy in HeLa cells (Figure 1, A–E). Colocalization between M6PRs and the organelle markers was quantified using Mander’s colocalization coefficients M1 and M2. Because M6PRs do not localize to the mitochondria, colocalization of M6PRs with mitochondria stained with MitoTracker dye was used as a negative control. As M6PRs are continuously trafficked from the TGN to early and late endosomes and back to the TGN, high colocalization of both CD-M6PRs and CI-M6PRs with the TGN (marked by TGOLN2), early endosomes (marked by RAB5A), and late endosomes (marked by RAB7A and LAMP1) was observed. Substantial colocalization of CD and CI-M6PRs with recycling endosomes (marked by RAB11A) was observed owing to cycling of M6PRs between the endocytic vesicles and the plasma membrane. These observations are in agreement with previous studies, which have reported that ∼0.35–0.40 and ∼0.20–0.25 fractions of both the M6PRs colocalize with the trans-Golgi apparatus and various endocytic compartments, respectively (Griffiths et al., 1988; Klumperman et al., 1993; Hirst et al., 1998; Tikkanen et al., 2000).

Development of constitutively active lysosomal hydrolase trafficking reporter

Although subcellular distribution of M6PRs provides some insights about lysosomal hydrolase trafficking, it is a surrogate readout to monitor the pathway, as it relies on the trafficking of the M6PRs rather than hydrolases themselves. Given the inadequacies of existing probes to monitor lysosomal hydrolase trafficking, including subcellular localization of M6PRs, a new probe with a simple and direct readout for hydrolase trafficking will be beneficial.

Inspired by existing ratiometric fluorescent probes, a novel lysosomal hydrolase trafficking reporter construct under a constitutive promoter (CMV), consisting of a lysosomal hydrolase (DNASE2 [deoxyribonuclease 2]), tandemly tagged with red (mCherry) and green (sfGFP) fluorescent proteins—“pCMV DNASE2-mCherry-sfGFP” was designed (Figure 2A). DNASE2, an established cargo of the M6P pathway (Evans and Aguilera, 2003; Čaval et al., 2019) and a previously used model for hydrolase trafficking (Ishii et al., 2019), was used as a representative lysosomal hydrolase. This hydrolase was chosen since its overexpression would not affect endogenous protein turnover in the cell. Tandemly tagged ratiometric fluorescent probes work based on differential acid sensitivity of red and green fluorescent proteins (Kimura et al., 2007). In the cellular compartments with higher pH, both red and green signals are visible, an overlap of which appears yellow (green⁺red⁺). Low pH in the lysosomal lumen causes quenching of the pH sensitive green fluorescence, while the pH insensitive red fluorescence remains unaffected, giving rise to green^quenched red⁺ signal, which appears red. When DNASE2-mCherry-sfGFP fusion protein is present in compartments with higher pH, such as ER, Golgi network, early endosomes, and recycling endosomes, both red and green signals are visible resulting in sfGFP⁺mCherry⁺ (apparent yellow) signal (Figure 2B). Entry of the fusion protein into the acidic lumen of the lysosome results in the sfGFP^quenchedmCherry⁺ (apparent red) signal. Because compartments with sfGFP^quenchedmCherry⁺ signal indicate the presence of hydrolases as well as acidic lumen simultaneously, they can be considered as a marker for active lysosomes. A change in the percentage of sfGFP^quenchedmCherry⁺ signal in the cell would indicate modulation of the hydrolase trafficking flux (Figure 2C). Higher and lower percentage of sfGFP^quenchedmCherry⁺ signal as compared with the steady state would indicate high and low hydrolase trafficking flux, respectively.

To confirm the expression of fusion protein “DNASE2-mCherry-sfGFP,” HeLa cells transfected with the reporter were subjected to immunoblotting. Immunoblot analysis of the lysates showed intact fusion protein of expected molecular weight (43 kDa [DNASE2] + 28 kDa [mCherry] + 28 kDa [sfGFP] = 99kDa) when probed with αGFP and αRFP antibodies (Supplemental Figure S1).

To validate M6P pathway–mediated trafficking of the reporter, we measured colocalization of the reporter with CD-M6PR and CI-M6PRs (Figure 2, D and E). HeLa cells transfected with the reporter were immune-stained for CD-M6PR, CI-M6PR, and COXIV. mCherry signal of the reporter was used for quantitation. Because lysosomal hydrolases do not localize to mitochondria, mitochondrial marker COXIV was used as a negative control. Substantial colocalization of the reporter was observed with both the M6PRs suggesting that the reporter trafficking is indeed mediated by M6P pathway.

Next, intracellular pH sensitivity of the reporter was validated by generating intracellular pH calibration curve (Figure 2, F–H). HeLa cells transfected with the reporter were incubated in potassium-phosphate buffer of varied pH (4, 4.5, 5, 5.5, 6, 6.5, 7, and 7.5) containing nigericin for 5 min followed by imaging in live-cell conditions according to the protocol described in the literature (Webb et al., 2021). Nigericin is an ionophore that equilibrates pH across membranes (Grillo-Hill et al., 2014; Gonzales and Canton, 2023). The ratio of sfGFP/mCherry fluorescence intensity per cell was recorded. The reporter showed sequential increase in the sfGFP/mCherry ratio from pH 4 to 7.5 (Figure 2G). The pKa value for the probe was calculated as described previously (Hoffmann and Kosegarten, 1995; Chin et al., 2021) (Figure 2H). pKa value of the reporter was found to be ∼6.1, which is close to the pKa value of sfGFP (5.9) reported previously (Roberts et al., 2016; Stoddard and Rolland, 2019). This result indicates intracellular pH sensitivity of the reporter over a range of pH, relevant to the intracellular lysosomal hydrolase trafficking.

To test the ability of the reporter to detect perturbations in the hydrolase trafficking pathway, HeLa cells expressing the reporter were treated with bafilomycin A1 (BafA1) (Figure 2, I–M). BafA1, being an inhibitor of the lysosomal v-ATPase pump, hampers lysosomal acidification (Yoshimori et al., 1991; Tapper and Sundler, 1995). HeLa cells transfected with the reporter construct showed a small but significant increase in the number of sfGFP⁺mCherry⁺ puncta and decrease in the number of sfGFP^quenchedmCherry⁺ puncta indicating a partial block in the maturation of lysosomal hydrolases (Figure 2, J and K). To estimate the proportion of reporter delivered to lysosomes in control versus BafA1-treated cells, the percentage of sfGFP^quenchedmCherry⁺ puncta per cell was calculated (Figure 2L). As expected, BafA1 treatment resulted in a decrease in the percentage of sfGFP^quenchedmCherry⁺ puncta (Figure 2 M). However, the ratio of sfGFP/mCherry signal intensity was not altered significantly upon BafA1 treatment (Supplemental Figure S2B).

The effect of BafA1 on the sfGFP signal of the reporter was lesser than expected (small effect on the number of sfGFP^quenchedmCherry⁺ and sfGFP⁺mCherry⁺ puncta (Figure 2, J and K); no statistically significant effect on sfGFP intensity (Supplemental Figure S2). The intracellular distribution of the constitutive reporter was a combination of perinuclear blob-like structures (similar to Golgi) and punctate structures (similar to TGN-derived vesicles, endosomes, and lysosomes) (Figure 2, D and I). We speculated that the effect of BafA1 on sfGFP signal might be masked by reporter present in relatively less acidic Golgi apparatus. To delineate the pH sensitivity of the reporter across the trajectory of M6P pathway, we measured the ratio of sfGFP/mCherry signal intensity per cell in each of the following compartments, in the presence/absence of BafA1: cis-Golgi (marked by GM130), trans-Golgi (marked by TGOLN2) and lysosomes (marked by LAMP1) (Supplemental Figure S3, A–D). We observed that sfGFP/mCherry ratio is unaffected upon BafA1 treatment in the cis-Golgi, while it shows a significant increase in the trans-Golgi and lysosomes. Extent of the effect of BafA1 on sfGFP/mCherry ratio was found to be higher in lysosomes, as compared with the trans-Golgi (pH ∼6), owing to more acidic luminal pH of lysosomes (pH∼4.5–5.5) (Schapiro and Grinstein, 2000; Paroutis et al., 2004; Steinberg et al., 2010). These results indicate that, in case of the constitutive reporter, the effect of BafA1 is detectable when thresholded for trans-Golgi and lysosomal compartments. However, at the whole-cell level, the effect of BafA1 on the sfGFP signal is masked due to the enrichment of the reporter in the Golgi compartments.

In summary, the constitutive reporter for hydrolase trafficking showed intracellular pH sensitivity across pH range relevant for M6P pathway (4–7.5) (Figure 2, F–H) and expected subcellular localization (Supplemental Figure S3; Figure 2 D and E). However, the response of the constitutive reporter to perturbation in the pathway induced by BafA1 was marginal (Figure 2, J and K; Supplemental Figure S2). Limited sensitivity of the constitutive reporter can possibly be attributed to the following factors: 1) Continuous biogenesis of the reporter might cause enrichment of the reporter in the Golgi compartments, 2) Continuous maturation of the reporter might cause transition of the reporter from sfGFP⁺mCherry⁺ to sfGFP^quenchedmCherry⁺ form even before BafA1 treatment. We speculated that these factors might dilute the effect of modulations in the pathway.

Inducible reporter enables temporal and stage-specific investigation of lysosomal hydrolase trafficking pathway

We hypothesized that, having a temporal control over the reporter expression might help to overcome the limitations associated with the constitutive reporter, by enabling regulated biogenesis and trafficking of the reporter to the lysosomes. To achieve this, the reporter was subcloned under a tetracycline-inducible promoter: “pTet-On DNASE2-mCherry-sfGFP” (Figure 3A). In this system, the biogenesis of the fusion protein in the cell starts only after treatment with doxycycline and stops when doxycycline is removed. To monitor the time kinetics of hydrolase trafficking using the inducible reporter, HeLa cells transfected with the reporter construct were treated with doxycycline for 2 h (pulse), followed by a chase of 0, 6, 12, 18, and 24 h. Due to the tetracycline-inducible promoter, a finite pool of reporter molecules is synthesized during the pulse period. Trafficking of this reporter pool over time can be monitored during the chase period (Figure 3B). A gradual decrease in the number of sfGFP⁺mCherry⁺ puncta and an increase in the number of sfGFP^quenchedmCherry⁺ puncta was observed over the time of chase, when monitored at 0, 6, 12, 18, and 24 h intervals (Figure 3, C–F). Approximately 3-fold increase in the percentage of sfGFP^quenchedmCherry⁺ puncta was observed at 24 h chase as compared with 0 h. The 24 h chase timepoint was selected for further experiments since it showed the highest percentage of sfGFP^quenchedmCherry⁺ puncta. Overall, the inducible reporter could recapitulate M6PR-dependent hydrolase trafficking pathway in a temporal manner.

Next, the subcellular distribution of the inducible reporter was mapped in a temporal manner (Figure 3, G and H). ER and cis-Golgi (starting point of the trajectory of lysosomal hydrolase trafficking), and lysosomes (endpoint of the trajectory of lysosomal hydrolase trafficking) were selected for the analysis. HeLa cells transfected with the inducible reporter were treated with doxycycline for 2 h (pulse) followed by 0 or 24 h chase. The cells were then immune-stained for KDEL (ER marker), GM130 (cis-Golgi marker), and LAMP1 (lysosomal marker). mCherry signal of the reporter was used for analysis as it remains stable across timepoints and organelles. At the 0 h chase timepoint, the reporter showed substantial colocalization with the ER network and blob-like perinuclear cis-Golgi structures, while localization to lysosomes was negligible (Figure 3, G and H). On the contrary, at 24 h chase timepoint, the reporter appeared punctate and showed high colocalization with lysosomes and very low colocalization with ER and cis-Golgi. These results indicate that the reporter faithfully follows the trajectory of the M6P pathway. Using such stage-specific organelle markers along with the temporal chase of the reporter, the stage at which M6P pathway flux is modulated in diseased state and by genetic or pharmacological agents can be identified.

Inducible reporter responds to changes in the hydrolase trafficking flux

Lysosomal hydrolase trafficking proceeds in a stepwise manner, wherein the hydrolases traverse through ER, Golgi network, early endosomes, late endosomes; finally reaching the lysosomes. Recycling of M6PRs from the late endosomes to the TGN is another critical step in the hydrolase trafficking pathway. Chemical agents that modulate specific stages of hydrolase trafficking pathway are known. The ability of the inducible reporter to track disturbances in the hydrolase trafficking pathway at different stages was investigated using some representative chemical modulators. HeLa cells expressing the reporter were subjected to a 2 h pulse of doxycycline followed by treatment with various chemical agents during the 24 h chase period (Figure 4, A–D). Brefeldin A (BFA), an inhibitor of ER to Golgi transport (Fujiwara et al., 1988; Lippincott-Schwartz et al., 1989), is previously reported to block the trafficking of lysosomal hydrolases (Oda and Nishimura, 1989). BFA treatment resulted in elevated sfGFP⁺ mCherry⁺ puncta and depleted sfGFP^quenched mCherry⁺ puncta as compared with the control (Figure 4, A–C). BFA treatment also caused decrease in the colocalization of the reporter with LAMP1, indicating block in delivery of the reporter to lysosomes (Supplemental Figure S5, A and B). Nocodazole, an inhibitor of microtubule polymerization (Vasquez et al., 1997; Smurova et al., 2008), has been reported to disrupt the trafficking of lysosomal hydrolases from the Golgi apparatus to the lysosomal compartments (Scheel et al., 1990). As anticipated, nocodazole treatment resulted in increased sfGFP⁺mCherry⁺ puncta and decreased sfGFP^quenched mCherry⁺ puncta (Figure 4, A–C). The last step of hydrolase trafficking pathway, endosomes to lysosomes transport or maturation, was investigated using BafA1 and chloroquine (CQ). BafA1 blocks the influx of H⁺ ions into the lysosomal lumen, rendering them inactive (Yoshimori et al., 1991). CQ being a weak base, accumulates in the lysosomal acidic lumen, thereby increasing the intravesicular pH (Homewood et al., 1972; Oda et al., 1991; Yoshimori et al., 1991; Tapper and Sundler, 1995; Mauthe et al., 2018). Some studies have also reported perturbation of anterograde transport by BafA1 and CQ (Clague et al., 1994; van Deurs et al., 1996; Bayer et al., 1998; Mousavi et al., 2001; Mauthe et al., 2018). Treatment with BafA1 and CQ caused accumulation of sfGFP⁺mCherry⁺ puncta, indicative of nonacidic compartments and concurrent decrease in sfGFP^quenchedmCherry⁺ puncta (Figure 4, A–C). Notably, change in the number of sfGFP⁺mCherry⁺ and sfGFP^quenchedmCherry⁺ puncta upon BafA1 treatment was more prominent in case of the inducible reporter as compared with constitutive reporter (Figure 2, I–M; Figure 4, A–D). Additionally, BafA1 treatment showed significant increase in the ratio of sfGFP/mCherry intensities at 12 as well as 24 h chase timepoints as compared with control in case of inducible reporter, as opposed to the constitutive reporter (Supplemental Figure 2B and S4C). These observations indicate the superior efficiency of inducible reporter in sensing modulations in the pathway. Moreover, the ratio of sfGFP/mCherry intensity in the lysosomes (marked by LAMP1) was lower in the control cells as compared with BafA1-treated cells (Supplemental Figure S5C). Treatment with amiodarone (Ami), a cationic amphiphilic drug known to cause redistribution of M6PRs from the TGN to endosomes (Gaffet et al., 1997; Ikeda et al., 2008; McGourty et al., 2012), also hampered hydrolase trafficking as indicated by increased sfGFP⁺mCherry⁺ puncta and decreased sfGFP^quenchedmCherry⁺ puncta number as compared with the control. Inhibition of the pathway by all these chemical agents resulted in a striking decrease in the percentage of sfGFP^quenched mCherry⁺ puncta as compared with the control, indicating that the reporter reliably reflects disturbances in the lysosomal hydrolase trafficking pathway at various stages.

FIGURE 4: — Inducible reporter responds to changes in hydrolase trafficking flux. (A) Representative microscopy images of HeLa cells showing the effect of known chemical modulators of hydrolase trafficking on the inducible reporter. HeLa cells transfected with the inducible reporter were treated with doxycycline for 2 h (pulse) followed by chase for 24 h in the presence/absence of the compounds. UT: untreated, DMSO: Dimethyl sulfoxide as solvent control, BFA: Brefeldin A, Noco: Nocodazole, BafA1: Bafilomycin A1, CQ: Chloroquine, Ami: Amiodarone. Scale bar, 10 μm. (B–D) Quantitation of the effect of compound treatment on the reporter (n ≥ 25, N = 3). (B) Number of sfGFP⁺mCherry⁺ reporter puncta per cell. ns, nonsignificant; ****, p < 0.0001, Kruskal–Wallis test with Dunn’s multiple comparisons test with UTs as control group. Mean ± SEM. (C) Number of sfGFP^quenchedmCherry⁺ reporter puncta per cell. ns, nonsignificant; ***, p < 0.001; ****, p < 0.0001, Kruskal-Wallis test with Dunn’s multiple comparisons test with UT as control group. (D) Percentage of sfGFP^quenchedmCherry⁺ reporter puncta per cell. ns, nonsignificant; ****, p < 0.0001, one-way ANOVA with Dunnett’s multiple comparisons test with UT as control group. Mean ± SEM. (E) Representative microscopy images of HeLa cells showing the effect of puromycin-induced intracellular protein aggregates on the inducible reporter. HeLa cells transfected with the inducible reporter were treated with doxycycline for 2 h (pulse) followed by 24 h chase with/without puromycin treatment and immunostaining for p62. Scale bar, 10 μm. (F) Quantitation of the size of p62 puncta upon puromycin treatment. A total of 500 puncta from 75 cells were quantified (n = 25 cells, N = 3 experiments). ****, p < 0.0001, Kolmogorov–Smirnov test. Mean ± SEM. (G–I) Quantitation of the effect of puromycin-induced intracellular protein aggregates on the reporter (n ≥ 25, N = 3). ****, p < 0.0001; Mann–Whitney test. Mean ± SEM. (G) Number of sfGFP⁺mCherry⁺ reporter puncta per cell. (H) Number of sfGFP^quenchedmCherry⁺ reporter puncta per cell. (I) Percentage of sfGFP^quenched mCherry⁺ reporter puncta per cell.

Defects in the endolysosomal system are a hallmark of neurodegenerative disorders. Accumulation of aggregated proteins in these conditions has also been proposed to interrupt the lysosomal hydrolase trafficking pathway, thereby facilitating further accumulation of aggregated proteins. To investigate whether the inducible reporter can detect disturbance in the hydrolase trafficking pathway induced by intracellular protein aggregates, we used puromycin-induced aggregation in HeLa cells as a model system. Puromycin is known to induce translational arrest, resulting in the formation of misfolded protein aggregates, termed as defective ribosomal products (Salomons et al., 2009; Lacsina et al., 2012; Aviner, 2020). HeLa cells expressing the inducible reporter were subjected to doxycycline pulse for 2 h followed by puromycin treatment during 24 h chase period (Figure 4, E–I). Induction of protein aggregates formed upon puromycin treatment was validated by immunostaining for p62, an adaptor protein routinely used to mark intracellular protein aggregates. A significantly larger size of p62 puncta in puromycin-treated cells as compared with the control cells confirmed the induction of intracellular protein aggregates (Figure 4, E and F). HeLa cells transfected with the reporter construct showed a notable increase in the number of sfGFP⁺mCherry⁺ puncta and a depleted number and percentage of sfGFP^quenchedmCherry⁺ puncta upon puromycin treatment compared with control, suggesting that the reporter can detect disruption in the lysosomal hydrolase trafficking pathway induced by intracellular protein aggregates (Figure 4, E–I).

Inducible reporter can track modulations in the hydrolase trafficking flux across cell types

The performance of the inducible reporter was tested across cells from multiple lineages to evaluate the versatility of the reporter assay and to assess the similarities and differences in the hydrolase trafficking pathway across cell lines. HEK293, HCT116, and PANC-1 cell lines were used for this purpose, apart from HeLa cells used in the previous experiments. HCT116 (intestinal epithelial cell line) and PANC-1 (pancreatic ductal adenocarcinoma cell line) cell lines were particularly chosen given the relevance of the hydrolase trafficking pathway in intestinal pathogenic infections and cancer.

HEK293, HCT116, and PANC-1 cells transfected with the inducible reporter were subjected to a 2 h pulse of doxycycline, followed by a 24 h chase with or without BafA1 treatment. Concurrent with the earlier results in HeLa cells, all the three tested cell lines showed an increase in the number of sfGFP⁺mCherry⁺ puncta and a decrease in the number and percentage of sfGFP^quenchedmCherry⁺ puncta upon BafA1 treatment as compared with control cells (Figure 5, A–E). Interestingly, there were specific differences in the responses of the cell lines tested. At the 24 h chase timepoint, the number of reporter puncta in HEK293 and HCT116 cells were similar to that of HeLa cells, while PANC-1 cells were found to have ∼10-fold higher number of reporter puncta (Figure 5, A–D). This aligns with a previous report that observed a higher number of lysosomes in pancreatic cancer cells (Perera et al., 2015). Moreover, PANC-1 cells showed lower percentage (∼40%) of sfGFP^quenched mCherry⁺ puncta per cell at the 24 h chase timepoint, as compared with other cell lines tested (∼85–90%), suggesting a slower rate of maturation of hydrolases in PANC-1 cells (Figure 5E). In addition, PANC-1 cells exhibited a more drastic depletion in the percentage of sfGFP^quenchedmCherry⁺ puncta upon BafA1 treatment (∼8-fold) as compared with other cell lines tested (∼5-fold) (Figure 5E). These results indicate that the inducible reporter is not only competent for measuring hydrolase trafficking flux of cells from different lineages but also serves to identify similarities and differences in the hydrolase trafficking flux across cell types.

FIGURE 5: — Inducible reporter can track modulations in the hydrolase trafficking flux across cell types. (A) Representative microscopy images of HEK293, HCT116, and PANC-1 cells transfected with the inducible reporter, treated with doxycycline for 2 h (pulse) followed by 24 h chase with/without BafA1. Scale bar, 10 μm. (B–D) Quantitation of the effect of BafA1 treatment on the reporter across cell types. Number of sfGFP⁺mCherry⁺ reporter puncta per cell and number of sfGFP^quenchedmCherry⁺ reporter puncta per cell was quantitated for each cell type. *, p < 0.05; ****, p < 0.0001; Kruskal–Wallis test with Dunn’s multiple comparisons test, mean ± SEM. (B) HEK293 cells (n ≥ 25, N = 3). (C) HCT116 cells. (n ≥ 25, N = 3). (D) PANC-1 cells. (n ≥ 18, N = 3). (E) Quantitation of the percentage of sfGFP^quenchedmCherry⁺ reporter puncta per cell across HeLa, HEK293, HCT116, and PANC-1 cells. (HeLa cells data same as in Figure 4D). ****, p < 0.0001, one-way ANOVA with Sidak’s multiple comparisons test. Mean ± SEM.

DISCUSSION

Optimal trafficking of lysosomal hydrolases to lysosomes via the M6P pathway is crucial for the maintenance of functional lysosomes. Although the molecular machinery of this pathway is thought to be well understood, the recent unexpected discovery of a previously unidentified core component of the pathway has highlighted that the regulatory mechanisms of the pathway are still underexplored (Pechincha et al., 2022; Richards et al., 2022; Zhang et al., 2022). Emerging evidence also points to the critical role of the M6P pathway in several disease pathologies including lysosomal storage disorders, neurodegeneration and intracellular infections (Matrone et al., 2016; Mazzulli et al., 2016; Carosi et al., 2021; Richards et al., 2022; Bird et al., 2023; Calcagni’ et al., 2023). The exigency for exploring the regulatory mechanisms of the pathway in physiological and pathophysiological states necessitates the availability of an efficient reporter to specifically track hydrolase trafficking flux.

Classical tools to measure the flux of lysosomal hydrolase trafficking, such as fluorography and immunoblotting of hydrolases, rely on differential glycosylation patterns and molecular weights of immature and mature forms of hydrolases (Hasilik and Neufeld, 1980; Oda and Nishimura, 1989; Kornfeld, 1990; Tavares and daSilva, 2017; Cuddy and Mazzulli, 2021). However, these tools do not allow for the visualization of hydrolases in different subcellular compartments, and thus, do not facilitate the understanding of the dynamics of various stages of hydrolase trafficking. Monitoring subcellular distribution of M6PRs by quantifying colocalization of M6PRs with various intracellular compartmental markers is another routinely used readout for hydrolase trafficking (Waguri et al., 2003; Seaman, 2004; Anitei et al., 2014; Breusegem and Seaman, 2014). However, this technique is an indirect readout, since it relies on the localization of the hydrolase receptors, rather than hydrolases themselves. Besides, fluorography, immunoblotting, and M6PR localization analysis are laborious and time-intensive methods and thus are inconvenient for large-scale screens.

There have been attempts to develop more efficient tools to study lysosomal hydrolase trafficking. Ishii et al. devised fluorescent probes consisting of lysosomal hydrolases and lysosomal membrane proteins tagged with sfGFP-T2A-mCherry aimed to study overall lysosomal homeostasis as well as the trafficking of lysosomal proteins (Ishii et al., 2019). During translation of the probe, sfGFP-tagged lysosomal protein is transported to the lysosomes, while free mCherry is released into the cytosol due to cotranslational cleavage of the nascent polypeptide chain at the T2A site. Optimum lysosomal acidic pH causes quenching of acid sensitive sfGFP signal while the mCherry signal remains unaltered. The ratio of mCherry to sfGFP fluorescent signal can detect perturbations in the trafficking of lysosomal hydrolases and lysosomal membrane proteins. Although release of free mCherry due to T2A cleavage site provides an internal control for the probe expression, it disables direct visualization of hydrolases present in the lysosomes. Moreover, dependence on T2A cleavage efficiency may decrease the sensitivity of the probe. Importantly, the assay design does not support time- and stage-dependent tracking of hydrolases.

We specifically aimed to spatiotemporally visualize and quantitate the trafficking of lysosomal hydrolases from the ER to lysosomes. Inspired by the routinely used tandemly tagged autophagy reporter (Kimura et al., 2007) and the probe developed by Ishii et al. (Ishii et al., 2019), we developed a new reporter consisting of a lysosomal hydrolase tandemly tagged with mCherry and sfGFP—“pCMV DNASE2-mCherry-sfGFP.” The reporter appears yellow (sfGFP⁺mCherry⁺) when present in the ER, Golgi network, or pre-lysosomal endocytic compartments, whereas it appears red (sfGFP^quenchedmCherry⁺) when present in acidic lysosomes. Unlike the probe devised by Ishii et al., it allows visualization of hydrolases present in acidic lysosomes along with all the other cellular compartments. We illustrated that the reporter showed a sequential increase in the ratio of sfGFP/mCherry intensity across intracellular pH ranging from 4 to 7.5, that is relevant for lysosomal hydrolase trafficking. Further, the reporter showed substantial colocalization with cis-Golgi, trans-Golgi, CD-M6PRs, CI-M6PRs, and lysosomes, validating trafficking of the reporter across the trajectory of M6P pathway. However, continuous biogenesis and maturation of the reporter, owing to its constitutively active nature, was speculated to limit its sensitivity in tracking subtle modulations in the hydrolase trafficking pathway. Moreover, it was not suitable to follow the kinetics of this process.

To bypass these constraints of our constitutive reporter assay, biogenesis of a limited pool of the tagged hydrolases (pulse) and tracking the transport of the pool over time (chase) is desirable. Previously used pulse-chase strategies to monitor intracellular trafficking are cycloheximide chase assay and Halo tags (Merrill et al., 2019; Rudinskiy et al., 2022; Yim et al., 2022; Zhang et al., 2022). Cycloheximide chase assay involves a block in the global protein synthesis induced by cycloheximide which can have off-target effects on the trafficking flux under study. Halo-GFP tag has been previously used to study autophagic flux in mammalian cells (Yim et al., 2022). A recent study that introduced a Halo-GFP reporter for probing protein and organelle cargo delivery to lysosomes has proposed the potential use of the reporter for monitoring lysosomal hydrolase trafficking (Rudinskiy et al., 2022). However, this technique may not be suitable for large-scale screening, given the requirement of expensive cell permeable fluorescent ligands and the need for careful optimization of the pulse-chase protocol.

To circumvent the limitations of these prevalent pulse-chase strategies, we used a tetracycline-inducible promoter system to temporally control the expression of our reporter. Pulse-chase analysis of hydrolase trafficking using our inducible reporter “pTet-On DNASE2-mCherry-sfGFP” allowed temporal and stage-specific investigation of the pathway. The inducible reporter also showed higher sensitivity to perturbations in hydrolase trafficking induced by a lysosomal proton pump inhibitor (BafA1) as compared with the constitutive reporter. Further, we demonstrated that our inducible reporter can successfully detect changes in hydrolase trafficking at various stages of the pathway upon treatment with known chemical modulators of the pathway. Moreover, the reporter could detect perturbations in hydrolase trafficking due to puromycin-induced intracellular aggregates. This provides evidence for the utility of the reporter in detecting disturbances in the trafficking of lysosomal hydrolases in disease conditions. “DNASE2” part of the reporter dictates its trafficking along the M6P pathway, “mCherry-sfGFP” part acts as a pH detection module, while Tet-On promoter of the inducible reporter facilitates temporal control over reporter expression. These three components together make the reporter capable of reliably tracking the M6P pathway in a temporal and stage-specific manner in steady and perturbed states of the pathway. If coupled with markers for intracellular compartments, the exact stage of trafficking modulation in diseased states can be deciphered.

Uncompromised reporter functionality in a variety of cell lines exhibited robustness and versatility of the reporter. Monitoring hydrolase trafficking flux using the reporter across different cell lines also revealed interesting cell lineage–specific differences. In particular, the pancreatic cancer cell line PANC-1, exhibited a 10-fold higher number of hydrolase-containing vesicles, a delayed rate of delivery of hydrolases to the lysosomes, and responded more drastically to perturbations in the flux as compared with other cell lines tested.

Our results indicate that at 0 h chase, approximately 20% of the reporter is present in acidic lysosomal compartments. As the trafficking proceeds over time, more of the reporter molecules move from less acidic cellular compartments to the more acidic lysosomes. Approximately 50% and 80% hydrolases were delivered to the lysosomes by 12 h and 24 h chase timepoints, respectively. Previous radiolabelling-based pulse-chase studies of lysosomal proteases (cathepsins D and H) reported that complete trafficking of the cathepsins takes ∼2 to 9 h (Oda and Nishimura, 1989; Scheel et al. 1990; Rijnboutt et al. 1992). Our reporter system, however, displayed a comparatively longer timeframe for complete trafficking of our tagged DNASE2 to lysosomes. The relatively delayed delivery of DNASE2 reporter viz-a-viz the cathepsins, may be explained as follows: first, the amount of DNASE2 reporter delivered to lysosomes at early timepoints may be lesser than the limit of detection for the assay. Second, as the time kinetics of endogenous DNASE2 delivery has not been studied till now, there is a possibility that the delivery of DNASE2 indeed takes longer time than that of the previously studied cathepsins. Third, addition of tandem mCherry-sfGFP tag delays the transport of DNASE2. To test these possibilities, temporal monitoring of the trafficking of endogenous DNASE2 enzyme is necessary.

In conclusion, the inducible hydrolase trafficking reporter is a simple, cost-effective, and useful tool to study lysosomal hydrolase trafficking flux in mammalian cells. We have shown that hydrolase trafficking can be efficiently studied using this reporter by fluorescence microscopy. The assay can potentially be optimized for flow cytometric readout as well. DNASE2 has been used as a model lysosomal hydrolase in this study. The simple and utilitarian design of the reporter allows for replacing DNASE2 with a disease-specific, wild-type, or mutant hydrolase, which can serve to dissect disease-specific mechanistic underpinnings of the pathway. The lentiviral design of the reporter allows for preparation of stable cell lines with the reporter. Growing evidence regarding the role of lysosomal hydrolase trafficking defects in various diseases has created the need for identification of genetic and pharmacological modulators of the pathway, which can rescue the diseased states. Our assay can be optimized for large-scale screens required for this purpose.

MATERIALS AND METHODS

Chemicals and reagents

Trypsin-EDTA (ethylenediaminetetraacetic acid) (59418C), doxycycline hydrochloride (D3447), nocodazole (M1404), CQ (C6628), brefeldin A (B6542), amiodarone hydrochloride (A8423), BafA1 (B1793), Nigericin sodium salt Ready Made Solution (SML1779) were purchased from Sigma-Aldrich. LysoTracker Deep Red (L12492) and Mito Tracker Orange CMTMRos (M7510) were purchased from Thermo Fisher Scientific.

Antibodies

Following is a list of antibodies used in the study:

Antibody Name and Catalogue No.	Company
IGF2R (MA1-066), TGOLN2 (PA5-23068), KDEL (PA1-013)	Invitrogen
M6PR (22d4)	Developmental Studies Hybridoma Bank
GFP	Roche Applied Sciences
SQSTM1/p62 (ab56416),	Abcam
RFP (6G6)	ChromoTek
β-tubulin (4466S), GM130 (D6B1) (12480), RAB5 (3547S), LAMP1 (9091P)	Cell Signaling Technology
Anti-mouse IgG conjugated with horseradish peroxidase (HRP) (1721011), anti-rabbit IgG (1706515) conjugated with HRP	Bio-Rad
Atto 633 (goat anti-rabbit IgG; 41176), Atto 488 (goat anti-rabbit IgG; 41057	Sigma-Aldrich

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Plasmid constructs

Plasmids used in this study include CD-MPR-CFP (a kind gift from Juan S. Bonifacino, NIH, USA), pCW DNASE2α-sfGFP-T2A-mCherry and pCW mCherry-sfGFP (a kind gift from Eisuke Itakura, Chiba University, Japan), and pcDNA3.1(+) (a kind gift from Ranga Udaykumar, JNCASR, India). mCherry-TGNP-N-10 (Addgene plasmid # 55145, deposited by Michael Davidson), mRFP-Rab5 (Addgene plasmid # 14437, deposited by Ari Helenius), mCh-Rab7A (Addgene plasmid # 61804, deposited by Gia Voeltz), Lamp1-RFP (Addgene plasmid # 1817, deposited by Walther Mothes) and dsRed-Rab11 WT (Addgene plasmid # 12679, deposited by Richard Pagano) were purchased from Addgene (Supplementary Table S1).

Molecular cloning

mCherry-sfGFP and DNASE2 fragments were sequentially subcloned into pcDNA3.1(+) backbone to generate pCMV DNASE2-mCherry-sfGFP as follows: mCherry-sfGFP fragment was amplified using pCW mcherry-sfGFP as a template by PCR with primers oMB1 and oMB2. The PCR amplicon was digested with restriction enzymes XbaI and ApaI and subcloned into pcDNA3.1(+) using the same restriction sites to generate pCMV mCherry-sfGFP (pMB1) plasmid. DNASE2 fragment was amplified using pCW DNASE2α-sfGFP-T2A-mCherry plasmid (pCW57.1 Lysosomal-METRIQ, Addgene #135401) as template by PCR with primers oMB3 and oMB4. The PCR amplicon was digested with restriction enzymes AflII and XbaI and subcloned into pMB1 to generate pCMV DNASE2-mCherry-sfGFP (pMB2). DNASE2 fragment was excised from pMB2 with restriction enzymes NheI and KspAI and inserted in pCW mcherry-sfGFP using the same restriction sites to generate pTet-On DNASE2-mCherry-sfGFP (pMB3) (Supplementary Tables S1 and S2).

Cell culture

HeLa, HEK293, HCT116, and PANC-1 cells were grown in DMEM (Sigma-Aldrich, 5648) supplemented with 10% FBS (Life Technologies, 10270-106), 100 units/ml penicillin, and streptomycin (Life Technologies, 15140-122) and 3.7 g/l sodium bicarbonate (Sigma-Aldrich, S5761), and were maintained in 5% CO₂ at 37°C.

Transfection of mammalian cells

Coverslips with cells seeded on them at 50% confluency were transfected with plasmid constructs using lipofectamine 2000 (Invitrogen, 11668019) following the manufacturer’s protocol. After 24 h of transfection, compound treatment was performed for the necessary amounts of time (as indicated), and cells were subsequently fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148). Staining was performed using fluorescence antibodies and the coverslips were mounted using Vectashield Antifade Mounting Medium with DAPI (Vector laboratories, H1000).

Immunofluorescence staining

Coverslips with cells seeded and attached were transfected with plasmid constructs and/or given compound treatments. For immunofluorescence, cells were permeabilized using 0.25% Triton X-100 (HiMedia Laboratories, MB031), followed by overnight incubation at 4°C with the primary antibodies (as indicated) and an hour of secondary antibody incubation at room temperature. Vectashield antifade reagent with DAPI (Vector laboratories, H-1000) was then used to mount the coverslips.

Immunoblotting

A total of 6-well plates seeded with cells at appropriate confluency were transfected and/or treated with compounds as indicated. For immunoblotting, cells were collected in Laemmli buffer (10% SDS [HiMedia Laboratories, GRM886], 0.05% bromophenol blue [Thermo Fisher Scientific, 115-39-90], 2M Tris-HCl [Merck, 1.93315.0521], 10 mM DTT [Thermo Fisher Scientific, R0862], 20% glycerol [Merck, 1.07051.0521], pH 6.8) using cell scraper and boiled for 10 min at 99^°C. After samples underwent SDS–PAGE electrophoresis, they were transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 1,620,177). The gel documentation system (G-box, Chemi XT 4, Syngene, USA) was used to acquire the image while the enhanced chemiluminescence substrate (Clarity Bio-Rad, 170-5061) was used to obtain the band signals following overnight primary antibody incubation at 4°C and HRP-conjugated secondary antibody incubation for 1 h at room temperature.

Fluorescent microscopy

Images were captured with an Olympus 60X/1.42 NA objective on the DV Elite fluorescence microscope (Leica Microsystems) using the DAPI, FITC, TRITC, and Cy5 filters for the following figures: Figure 1, B and C; Figure 2, D, F, and I; Figure 3G; Figure 4E; Supplemental Figure S3A; Supplemental Figure S4A; Supplemental Figure S5A. The pictures were deconvolved postacquisition using DeltaVision SoftWorx software. Images were acquired on the CQ1-Yokogawa Benchtop High-Content Analysis System epifluorescence microscope using DAPI, FITC, TRITC, and Cy5 lasers without deconvolution for the following Figures: Figure 3C, Figure 4A, Figure 5A. The number and size of Z sections, fluorescence intensity, and percentage transmittance were maintained constant for each experiment across treatments.

Image analysis

Details of image analysis procedures followed for each type of analysis using ImageJ are listed below:

Colocalization analysis using Manders’ coefficients M1 and M2

Mander’s coefficients were calculated with JaCoP Plugin in ImageJ using a customized semiautomated macro with “default” autothresholding for each image. Macro used for the analysis in Figure 1, D and E is included in Supplemental Material (Figure 1, D and E_macro).

Mean intensity of sfGFP and mCherry signals

The entire cell was selected as region of interest (ROI). Background signals in sfGFP and mCherry channels were subtracted (“Process>subtract background”; [rolling ball radius = 50 pixels]) across all datasets. Intensity of signal in the ROI for sfGFP and mCherry channels was measured without any thresholding (“Analyze>Measure>Mean intensity”). This protocol was followed for all the datasets using an automated macro enabling unbiased analysis (Supplemental Figure S2A_macro in Supplemental Material).

Ratio of sfGFP/mCherry intensity per cell

The ratio of sfGFP/mCherry signal intensity was calculated based on mean intensity values of sfGFP and mCherry.

Number of sfGFP ⁺mCherry ⁺ and sfGFP ^quenchedmCherry ⁺ puncta per cell

Number of sfGFP⁺mCherry⁺ puncta
- A window showing colocalization between sfGFP and mCherry channels was obtained using “colocalization plugin.” (“MaxEntropy” autothresholding algorithm used [“Image>Adjust>Threshold” tool] to threshold sfGFP and mCherry channels).
- The colocalization window was autothresholded using “MaxEntropy” algorithm. The number of sfGFP⁺mCherry⁺ puncta per cell in the colocalization window were counted using particle analysis tool.
Number of sfGFP^quenchedmCherry⁺ puncta
- The total number of mCherry⁺ puncta (sfGFP⁺mCherry⁺ + sfGFP^quenchedmCherry⁺ puncta) per cell were counted from mCherry channel using particle analysis tool (“MaxEntropy” autothresholding algorithm used). The number of sfGFP^quenchedmCherry⁺ puncta were calculated as follows:

Number of sfGFP^quenched mCherry⁺ puncta =

Total number of mCherry⁺ puncta − Number of sfGFP⁺mCherry⁺ puncta

Percentage of sfGFP ^quenchedmCherry ⁺ puncta per cell

Percentage of sfGFP^quenchedmCherry⁺ puncta per cell was calculated as follows:

Percentage of sfGFP^quenchedmCherry⁺ puncta =

Number of sfGFP^quenchedmCherry⁺ puncta/ (Number of sfGFP⁺mCherry⁺ puncta + Number of sfGFP^quenchedmCherry⁺ puncta) *100 (described in Figure 2L).

Percent reporter area colocalizing with the organelle per cell

Fraction of reporter area colocalizing with the organelle per cell was quantified using Mander’s coefficient (M2). This analysis was performed using JaCOP plugin in ImageJ by a customized semiautomated macro (similar to Supplemental Figure S3, F and G_macro in Supplemental Material). “Default” autothresholding was used for quantitation for all the datasets. Multiplying each of the “M1” values by 100 gave % reporter area colocalizing with the organelle.

Percent organelle area colocalizing with the reporter per cell

Fraction of organelle area colocalizing with sfGFP or mCherry counterparts of the reporter per cell was quantified using Mander’s coefficient (M1). This analysis was performed using JaCOP plugin in ImageJ by a customized macro. “Default” autothresholding was used for quantitation for all the datasets. Multiplying each of the “M1” values by 100 gave % organelle area occupied by the sfGFP and mCherry counterparts of the reporter. Macro used for the analysis of Supplemental Figure S3, F and G is included in Supplemental Material (Supplemental Figure S3, F and G_macro).

Ratio of sfGFP/mCherry intensity in indicated organelles per cell

The image was open in split channels. A single cell was selected as ROI. Background subtraction was performed for sfGFP and mCherry channels using Image>Process>Subtract background (rolling ball radius = 50 pixels) across all datasets. Threshold for organelle marker channels was set using “MaxEntropy” autothresholding algorithm across all datasets. The thresholded organelle area was added as an ROI (Edit>Selection>Create selection) and was overlayed onto sfGFP and mCherry channels. Mean intensity for sfGFP and mCherry channels in the thresholded organelle area was measured using Analyze>Measure function in ImageJ and the ratio of sfGFP/mCherry intensity in the organelle was calculated. This analysis was performed using a customized macro (Supplemental Figure S3, B–E_macro in Supplemental Material).

pH calibration curve

Cells were seeded at 50% confluency in glass bottom dishes and were allowed to attach overnight. A total of 24 h after transfection with CMV DNASE2-mCherry-sfGFP, the cells were incubated in potassium-phosphate buffer (50 mM potassium phosphate, 80 mM potassium chloride, 1 mM magnesium chloride) containing 20 μM nigericin free acid of varied pH (4, 4.5, 5, 5.5, 6, 6.5, 7, and 7.5) for 5 min at 37°C followed by imaging in live-cell conditions according to the protocol described in the literature (Webb et al., 2021). sfGFP and mCherry fluorescence values were obtained for each cell and the ratio of sfGFP/mCherry fluorescence intensities was calculated. log₁₀(sfGFP/mCherry) values were fit with a linear regression. The pKa value for the reporter was calculated using modified Henderson−Hasselbalch equation as described previously (Hoffmann and Kosegarten, 1995; Chin et al., 2021).

Pulse-chase analysis to track hydrolase trafficking

Cells were seeded at 50% confluency in 24-well plates and were allowed to attach overnight. A total of 24 h after transfection with Tet-On DNASE2-mCherry-sfGFP, the cells were treated with doxycycline (1 µg/ml) for 2 h (pulse), followed by two PBS washes and the addition of doxycycline-free media until chase timepoint. At each timepoint of chase (0, 6, 12, 18, and 24 h), the cells were fixed using paraformaldehyde or collected in Laemmli buffer as required.

Lysotracker and MitoTracker staining

Cells were seeded at 50% confluency and were allowed to attach overnight. The cells were transfected with plasmid constructs as required and stained with LysoTracker Deep Red (100 nM, 10 min) or MitoTracker Orange CMTMRos (200 nM, 10 min).

Compound treatment

Cells were seeded at 50% confluency in 24-well plates and were allowed to attach overnight. A total of 24 h posttransfection with Tet-On DNASE2-mCherry-sfGFP, cells were treated with doxycycline (1 µg/ml) for 2 h (pulse), followed by two PBS washes, and the cells were incubated in doxycycline-free media containing the compounds till the 24 h chase timepoint. The following compounds were used: nocodazole (0.75 µM), Brefeldin A (0.25 µg/ml), BafA1 (60 nM), amiodarone (8 µM), and CQ (20 µM). At the end of 24 h chase, the cells were fixed with paraformaldehyde and imaged.

Puromycin-induced aggregate formation

Cells were seeded at 50% confluency in 24-well plates and were allowed to attach overnight. A total of 24 h posttransfection with Tet-On DNASE2-mCherry-sfGFP, cells were treated with doxycycline (1 µg/ml) for 2 h (pulse) followed by two PBS washes. Next, the cells were incubated in doxycycline-free media with or without puromycin (2 µg/mL puromycin) till 24 h chase timepoint. Cells were fixed at the end of 24 h using 4% paraformaldehyde and stained for p62.

Statistical analysis and data representation

Unpaired two-tailed Student t tests, the Mann–Whitney test, one-way ANOVA, Kruskal–Wallis test, and various post-hoc tests as appropriate were used to determine the significance levels between the control and test groups. These tests were then followed by the appropriate multiple comparison test, where ns = nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 were used. The error bars indicate mean ± SEM. Plotting was done using GraphPad Prism 9.5.1 (Licensed), which was also used for all the statistical testing. “n” and ’N’ in the legends represent technical and experimental replicates, respectively. Three independent experiments were conducted for each observation made in this study.

Supplementary Material

mbc-35-mr6-s001.pdf^{(23.8MB, pdf)}

ACKNOWLEDGMENTS

We thank Eisuke Itakura (Chiba University, Japan) for sharing reagents and scientific information. We are extremely grateful to Subba Rao Gangi Setty (IISc, India) and Amit Tuli (CSIR IMTech, India) for scientific inputs. We acknowledge Juan S. Bonifacino, (NIH, USA) and Ranga Udaykumar (JNCASR, India) for sharing reagents. We are thankful to the members of the Autophagy Laboratory (JNCASR) and Aparna Hebbar for their inputs and critical review of the manuscript. We thank Bio Imaging facility at JNCASR and Varun M for technical support. We thank Rahul Dubey (Autophagy laboratory, JNCASR, India) and Yash Acharya (Antimicrobial research laboratory, JNCASR, India) for help in developing ImageJ macros for image analysis and scientific inputs about pH calibration curve experiments, respectively. Work done by L.E. was carried out at the Autophagy Laboratory (JNCASR). This work was supported by intramural funds from JNCASR to R.M., Science and Engineering Research Board (SERB) CRG grant (CRG/2019/004892), S. Ramachandran National Bioscience Award for Career Development by the Department of Biotechnology (DBT) (102/IFD/SAN/990/2021-2022), and the Centre for Marine Therapeutics (CMT) grant from the DoP (Department of Pharmaceuticals) and Department of Science and Technology (DST), Govt. of India (NIPERK/2023/CMT/01). M.B., A.N., I.M.A., and R.S. are thankful for doctoral fellowships by JNCASR. M.N. acknowledges RA fellowship by JNCASR.

Abbreviations used:

Ami: Amiodarone
BafA1: Bafilomycin A1
BFA: Brefeldin A
CD-M6PR: cation-dependent mannose-6-phosphate receptor
CI-M6PR: cation-independent mannose-6-phosphate receptor
CQ: chloroquine
DRiPs: defective ribosomal products
ER: endoplasmic reticulum
LIMP-2: lysosomal integral membrane protein-2
LSD: lysosomal storage disorder
Noco: nocodazole
TGN: trans-Golgi network.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E23-09-0344) on June 18, 2024.

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PERMALINK

A genetically-encoded fluorescence-based reporter to spatiotemporally investigate mannose-6-phosphate pathway

Mallika Bhat

Akshaya Nambiar

Lakshmi Edakkandiyil

Irine Maria Abraham

Ritoprova Sen

Mamta Negi

Ravi Manjithaya

Roles

Abstract

INTRODUCTION

FIGURE 1:

RESULTS

Tracking M6PRs: a routinely used method to monitor lysosomal hydrolase trafficking

Development of constitutively active lysosomal hydrolase trafficking reporter

FIGURE 2:

Inducible reporter enables temporal and stage-specific investigation of lysosomal hydrolase trafficking pathway

FIGURE 3:

Inducible reporter responds to changes in the hydrolase trafficking flux

FIGURE 4:

Inducible reporter can track modulations in the hydrolase trafficking flux across cell types

FIGURE 5:

DISCUSSION

MATERIALS AND METHODS

Chemicals and reagents

Antibodies

Plasmid constructs

Molecular cloning

Cell culture

Transfection of mammalian cells

Immunofluorescence staining

Immunoblotting

Fluorescent microscopy

Image analysis

Colocalization analysis using Manders’ coefficients M1 and M2

Mean intensity of sfGFP and mCherry signals

Ratio of sfGFP/mCherry intensity per cell

Number of sfGFP +mCherry + and sfGFP quenchedmCherry + puncta per cell

Percentage of sfGFP quenchedmCherry + puncta per cell

Percent reporter area colocalizing with the organelle per cell

Percent organelle area colocalizing with the reporter per cell

Ratio of sfGFP/mCherry intensity in indicated organelles per cell

pH calibration curve

Pulse-chase analysis to track hydrolase trafficking

Lysotracker and MitoTracker staining

Compound treatment

Puromycin-induced aggregate formation

Statistical analysis and data representation

Supplementary Material

ACKNOWLEDGMENTS

Abbreviations used:

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Number of sfGFP ⁺mCherry ⁺ and sfGFP ^quenchedmCherry ⁺ puncta per cell

Percentage of sfGFP ^quenchedmCherry ⁺ puncta per cell