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. 2012 Jun;69(12):2075-89.
doi: 10.1007/s00018-011-0913-1. Epub 2012 Jan 20.

Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

Kristiina Uusi-Rauva  1 , Aija Kyttälä, Rik van der Kant, Jouni Vesa, Kimmo Tanhuanpää, Jacques Neefjes, Vesa M Olkkonen, Anu Jalanko
Affiliations

Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

Kristiina Uusi-Rauva et al. Cell Mol Life Sci. 2012 Jun.

Abstract

CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3∆ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport.

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Figures

Fig. 1
Fig. 1
a–i The CLN3E295K mutant clusters late endosomes/lysosomes in a perinuclear region. Wild-type CLN3 (a) and the CLN3E295K mutant (d, g) were separately expressed in HeLa cells and analysed with endosomal/lysosomal markers by immunofluorescence microscopy. Contrary to the cells expressing wild-type CLN3 (b), expression of CLN3E295K induced a perinuclear clustering of LAMP-1-positive late endosomes and lysosomes (e), with no obvious effect on early endosomal EEA1-positive compartments (h). c, f and i represent the overlays of the immunofluorescence signals of indicated proteins. Scale bars 20 μm
Fig. 2
Fig. 2
a–m CLN3E295K affects compartmentalisation of EGFP-Rab7. HeLa cells transfected with EGFP-tagged Rab7 alone (a–c) or together with either wild-type CLN3 (d–h) or the CLN3E295K mutant (i–m) were processed for CLN3, CLN3E295K, EGFP-Rab7 and LAMP-1 immunofluorescence microscopy. Compared to the other conditions, EGFP-Rab7 in the CLN3E295K-transfected cells appeared to be more concentrated on CLN3E295K (l) and LAMP-1-positive (m) compartments. c′, g′, h′, l′ and m′ represent magnifications of co-localisation area in the corresponding images. Scale bars 20 μm
Fig. 3
Fig. 3
Quantification of the co-localisation between CLN3 and EGFP-Rab7, and EGFP-Rab7 and LAMP-1. To quantify the results visualised in Fig. 2, immunofluorescence images of HeLa cells tranfected for EGFP-Rab7 alone, CLN3 and EGFP-Rab7, or CLN3E295K and EGFP-Rab7 were analysed by measuring Pearson’s correlation value for co-localisation between indicated immunofluorescence stainings. Each data point represents mean ± SEM of 15 cells of one experiment. *P < 0.0005 (one-tailed Student’s t test)
Fig. 4
Fig. 4
The CLN3E295K-induced perinuclear clustering of lysosomes is dependent on intact microtubules and functional dynactin complex. HeLa cells expressing CLN3E295K were either treated with nocodazole (upper panel) or co-expressed with EGFP-p50dynamitin (lower panel) and processed for CLN3 and LAMP-1 immunofluorescence microscopy. In both cases, CLN3E295K and LAMP-1-positive perinuclear clusters were lost and instead, corresponding compartments were dispersed throughout the cytoplasm. Scale bars 10 μm
Fig. 5
Fig. 5
a, b Cytoplasmic acidification-driven outward movement of LAMP-1-positive late endosomes and lysosomes is defective in CLN3-silenced HeLa cells. CLN3 siRNA and control siRNA-transfected Hela cells were treated with Ringer’s solution pH 7.2 or with Ringer’s solution pH 6.9 and processed for LAMP-1 immunofluorescence microscopy. a Each analysed cell was classified in one of the three phenotypic categories based on the distribution of LAMP-1 immunofluorescence staining; normal, dispersed and extremely dispersed. b The percentage of each phenotype per condition is indicated. Microscopy analyses were carried single-blindly. Each data point represents mean ± SEM of two individual experiments, and at least 150 cells were scored for each condition in each experiment. *P < 0.04, **P = 0.066 (one-tailed Student’s t test). Scale bar 20 μm
Fig. 6
Fig. 6
CLN3 interacts with both minus and plus end-directed motor protein components. COS-1 cells transfected with or without CLN3 (untransfected cell control) were processed for immunoprecipitation with anti-CLN3 242–258 followed by western blotting analysis with antibodies against components of microtubular motor complexes: β-tubulin (55 kDa), p150 Glued (150 kDa), dynein intermediate chain (DIC, 74 kDa) and KIF3A subunit of the heterotrimeric kinesin-2 complex (80/85 kDa). All tested proteins co-immunoprecipitated specifically with CLN3 (43 kDa). Matrix control represents sample devoid of precipitating antibody. Load represents 2% of total protein present in immunoprecipitation
Fig. 7
Fig. 7
a, b Xpress-Rab7 and Xpress-RILP co-immunoprecipitate with CLN3. COS-1 cells transfected for Xpress-tagged Rab7 (a) or Xpress-tagged RILP (b) with or without CLN3 were processed for immunoprecipitation with anti-CLN3 242–258 followed by western blotting with anti-Xpress and anti-CLN3. Both Xpress-Rab7 (~30 kDa) (a) and Xpress-RILP (~60 kDa) (b) co-immunoprecipitated only from the cells that also expressed CLN3 (43 kDa). Matrix control represents sample devoid of precipitating antibody. Load represents 2% of total protein present in immunoprecipitation
Fig. 8
Fig. 8
a, b CLN3 interacts directly with active, GTP-bound Rab7 and RILP via different cytoplasmic domains. a Mammalian two-hybrid experiment with two cytoplasmic CLN3 domains and different nucleotide-bound forms of Rab7, a wild-type Rab7, a GTPase-deficient constitutively active Rab7Q67L mutant, and a dominant-negative Rab7T22N mutant. Only CLN3 1–40/Rab7wt and 1–40/Rab7Q67L two-hybrids resulted in higher CAT expression than the corresponding controls. Each data point represents mean of normalised expression levels of CAT reporter gene of three individual experiments ±sem (one experiment contained CLN3 + Rab7 hybrids only). *P < 0.05 and **P = 0.11 (one-tailed Student’s t test). b GST (control) or GST-tagged domains of CLN3, the N-terminus (amino acids 1–33) and cytoplasmic loop (232–280) purified by coupling to glutathione Sepharose were incubated with purified His6-tagged Rab7Q67L and/or RILP recombinant proteins. Figure shows the results of the subsequent western blot analyses with Rab7 and RILP specific antibodies. His6-Rab7Q67L (~23 kDa) was found to directly interact with the N-terminal domain of CLN3. His6-RILP (~55 kDa) interacts directly with the cytoplasmic loop of CLN3. Load represents the percentage of total protein used
Fig. 9
Fig. 9
a, b Loss of lysosomal CLN3 impairs the GTP/GDP cycle of Rab7. a To assess the possible effects of CLN3 deficiency on the GTP/GDP cycle of Rab7, fibroblasts from healthy control and from two juvenile CLN3 disease patients, carrying CLN3∆ex7-8/CLN3∆ex7-8 (homozygous) or CLN3E295K/CLN3∆ex7-8 (compound heterozygous) mutations, were transfected with EGFP-Rab7. The EGFP fluorescence of late endosomal/lysosomal compartments was bleached, and fluorescence recovery (shown relative to the starting value, y-axis) as a function of time (x-axis) was recorded. CLN3∆ex7-8 homozygous cells showed accelerated recovery rates of EGFP-Rab7 signal. Each data point represents mean ± SEM of five recordings from different cells. b Immunofluorescence images of different time points of recovering cells (shown for control and homozygous CLN3∆ex7-8/CLN3∆ex7-8 cells). The bleached area is depicted by a square. N indicates the nucleus
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