The Batten disease protein CLN3 is important for stress granules dynamics and translational activity.

The assembly of membrane-less organelles such as stress granules (SGs) is emerging as central in helping cells rapidly respond and adapt to stress. Following stress sensing, the resulting global translational shutoff leads to the condensation of stalled mRNAs and proteins into SGs. By reorganizing cytoplasmic contents, SGs can modulate RNA translation, biochemical reactions, and signaling cascades to promote survival until the stress is resolved. While mechanisms for SG disassembly are not widely understood, the resolution of SGs is important for maintaining cell viability and protein homeostasis. Mutations that lead to persistent or aberrant SGs are increasingly associated with neuropathology and a hallmark of several neurodegenerative diseases. Mutations in CLN3 are causative of juvenile neuronal ceroid lipofuscinosis, a rare neurodegenerative disease affecting children also known as Batten disease. CLN3 encodes a transmembrane lysosomal protein implicated in autophagy, endosomal trafficking, metabolism, and response to oxidative stress. Using a HeLa cell model lacking CLN3, we now show that CLN3KO is associated with an altered metabolic profile, reduced global translation, and altered stress signaling. Furthermore, loss of CLN3 function results in perturbations in SG dynamics, resulting in assembly and disassembly defects, and altered expression of the key SG nucleating factor G3BP1. With a growing interest in SG-modulating drugs for the treatment of neurodegenerative diseases, novel insights into the molecular basis of CLN3 Batten disease may reveal avenues for disease-modifying treatments for this debilitating childhood disease.

Mechanisms regulating the sorting of soluble lysosomal proteins.

Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer's disease, Parkinson's disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.

Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease).

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a family of neurodegenerative diseases that affect all age groups and ethnicities around the globe. At least a dozen NCL subtypes have been identified that are each linked to a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene. Mutations in CLN genes cause the accumulation of autofluorescent lipoprotein aggregates, called ceroid lipofuscin, in neurons and other cell types outside the central nervous system. The mechanisms regulating the accumulation of this material are not entirely known. The CLN genes encode cytosolic, lysosomal, and integral membrane proteins that are associated with a variety of cellular processes, and accumulated evidence suggests they participate in shared or convergent biological pathways. Research across a variety of non-mammalian and mammalian model systems clearly supports an effect of CLN gene mutations on autophagy, suggesting that autophagy plays an essential role in the development and progression of the NCLs. In this review, we summarize research linking the autophagy pathway to the NCLs to guide future work that further elucidates the contribution of altered autophagy to NCL pathology.

The first infrared beamline at the Middle East SESAME synchrotron facility.

SESAME (Synchrotron-light for Experimental Science and Applications in the Middle East) is the only synchrotron light facility in the Middle East and neighboring regions, officially opened in 2017. Among the identification and construction of the first operational beamlines, infrared spectromicroscopy was selected as one of the two beamlines to be opened to the general users' program (the so-called Day-1 beamlines). Being one of the most demanded techniques by various scientific communities in the Middle East, the beamline has been designed and implemented in the framework of a collaboration agreement with the French synchrotron facility, SOLEIL. The design, construction and initial performances of the IR beamline (D02-IR), nowadays operational, are reported.

CLN3, at the crossroads of endocytic trafficking.

The CLN3 gene was identified over two decades ago, but the primary function of the CLN3 protein remains unknown. Recessive inheritance of loss of function mutations in CLN3 are responsible for juvenile neuronal ceroid lipofuscinosis (Batten disease, or CLN3 disease), a fatal childhood onset neurodegenerative disease causing vision loss, seizures, progressive dementia, motor function loss and premature death. CLN3 is a multipass transmembrane protein that primarily localizes to endosomes and lysosomes. Defects in endocytosis, autophagy, and lysosomal function are common findings in CLN3-deficiency model systems. However, the molecular mechanisms underlying these defects have not yet been fully elucidated. In this mini-review, we will summarize the current understanding of the CLN3 protein interaction network and discuss how this knowledge is starting to delineate the molecular pathogenesis of CLN3 disease. Accumulating evidence strongly points towards CLN3 playing a role in regulation of the cytoskeleton and cytoskeletal associated proteins to tether cellular membranes, regulation of membrane complexes such as channels/transporters, and modulating the function of small GTPases to effectively mediate vesicular movement and membrane dynamics.

CLN5 and CLN3 function as a complex to regulate endolysosome function.

CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, neuronal ceroid lipofuscinosis (NCL). We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in NCL, RAB7A, and a subset of RAB7A effectors. Our data support a model where CLN3 and CLN5 function as an endolysosomal complex regulating various functions.

A microfluidic dosimetry cell to irradiate solutions with poorly penetrating radiations: a step towards online dosimetry for synchrotron beamlines.

Synchrotron radiation can induce sample damage, whether intended or not. In the case of sensitive samples, such as biological ones, modifications can be significant. To understand and predict the effects due to exposure, it is necessary to know the ionizing radiation dose deposited in the sample. In the case of aqueous samples, deleterious effects are mostly induced by the production of reactive oxygen species via water radiolysis. These species are therefore good indicators of the dose. Here the application of a microfluidic cell specifically optimized for low penetrating soft X-ray radiation is reported. Sodium benzoate was used as a fluorescent dosimeter thanks to its specific detection of hydroxyl radicals, a radiolytic product of water. Measurements at 1.28 keV led to the determination of a hydroxyl production yield, G(HO.), of 0.025 ±â€…0.004 µmol J-1. This result is in agreement with the literature and confirms the high linear energy transfer behavior of soft X-rays. An analysis of the important parameters of the microfluidic dosimetry cell, as well as their influences over dosimetry, is also reported.

FTIR micro-spectroscopy using synchrotron-based and thermal source-based radiation for probing live bacteria.

Fourier transform infrared (FTIR) spectroscopy has proven to be a non-invasive tool to analyse cells without the hurdle of employing exogenous dyes or probes. Nevertheless, the study of single live bacteria in their aqueous environment has long remained a big challenge, due to the strong infrared absorption of water and the small size of bacteria compared to the micron-range infrared wavelengths of the probing photons. To record infrared spectra of bacteria in an aqueous environment, at different spatial resolutions, two setups were developed. A custom-built attenuated total reflection inverted microscope was coupled to a synchrotron-based FTIR spectrometer, using a germanium hemisphere. With such a setup, a projected spot size of 1 × 1 µm2 was achieved, which allowed spectral acquisition at the single-cell level in the 1800-1300 cm-1 region. The second setup used a demountable liquid micro-chamber with a thermal source-powered FTIR microscope, in transmission geometry, for probing clusters of a few thousands of live cells in the mid-IR region (4000-975 cm-1). Both setups were applied for studying two strains of a model lactic acid bacterium exhibiting different cryo-resistances. The two approaches allowed the discrimination of both strains and revealed population heterogeneity among bacteria at different spatial resolutions. The multivariate analysis of spectra indicated that the cryo-sensitive cells presented the highest cell heterogeneity and the highest content of proteins with the α-helix structure. Furthermore, the results from clusters of bacterial cells evidenced phosphate and peptidoglycan vibrational bands associated with the cell envelope, as potential markers of resistance to environmental conditions. Graphical Abstract.

Cu-catalyzed click conjugation of cobalamin to a BODIPY-based fluorophore: A versatile tool to explore the cellular biology of vitamin B12.

The Cu-catalyzed click conjugation of an azide-functionalized vitamin B12 (cobalamin) and an alkyne-labeled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) led to the formation of a highly stable fluorescent BODIPY-labeled vitamin B12 (λex/λem = 495/508 nm). The formation of what has been identified as an iodine adduct of the conjugate was also observed as a side-product during this reaction and could be removed using HPLC. BODIPY-labeled vitamin B12 was characterized by NMR and HR-ESI-MS. In vitro studies on wild-type human fibroblasts indicated that BODIPY-labeled vitamin B12 could internalize in a manner similar to that of untagged vitamin B12. ATP-binding cassette sub-family D member 4 (ABCD4) is a lysosomal localized transporter required to export vitamin B12 from the lysosomal lumen to the cytosol. Mutations in this transporter result in the accumulation of vitamin B12 in lysosomes. In human fibroblasts harbouring a mutation in ABCD4, BODIPY-labeled vitamin B12 accumulated in the lumen of lysosomes. Our data suggests the potential use of BODIPY-labeled vitamin B12 to investigate the intracellular behavior of the vitamin in the context of disorders related to the abnormal cellular utilization of the vitamin. Moreover, results presented here demonstrate that click chemistry could be exploited for the conjugation of vitamin B12 to various other fluorophores.

A pressure-actuated flow cell for soft X-ray spectromicroscopy in liquid media.

We present and fully characterize a flow cell dedicated to imaging in liquid at the nanoscale. Its use as a routine sample environment for soft X-ray spectromicroscopy is demonstrated, in particular through the spectral analysis of inorganic particles in water. The care taken in delineating the fluidic pathways and the precision associated with pressure actuation ensure the efficiency of fluid renewal under the beam, which in turn guarantees a successful utilization of this microfluidic tool for in situ kinetic studies. The assembly of the described flow cell necessitates no sophisticated microfabrication and can be easily implemented in any laboratory. Furthermore, the design principles we relied on are transposable to all microscopies involving strongly absorbed radiation (e.g. X-ray, electron), as well as to all kinds of X-ray diffraction/scattering techniques.

CLN3 regulates endosomal function by modulating Rab7A-effector interactions.

Mutations in CLN3 are a cause of juvenile neuronal ceroid lipofuscinosis (JNCL), also known as Batten disease. Clinical manifestations include cognitive regression, progressive loss of vision and motor function, epileptic seizures and a significantly reduced lifespan. CLN3 localizes to endosomes and lysosomes, and has been implicated in intracellular trafficking and autophagy. However, the precise molecular function of CLN3 remains to be elucidated. Previous studies showed an interaction between CLN3 and Rab7A, a small GTPase that regulates several functions at late endosomes. We confirmed this interaction in live cells and found that CLN3 is required for the efficient endosome-to-TGN trafficking of the lysosomal sorting receptors because it regulates the Rab7A interaction with retromer. In cells lacking CLN3 or expressing CLN3 harbouring a disease-causing mutation, the lysosomal sorting receptors were degraded. We also demonstrated that CLN3 is required for the Rab7A-PLEKHM1 interaction, which is required for fusion of autophagosomes to lysosomes. Overall, our data provide a molecular explanation behind phenotypes observed in JNCL and give an indication of the pathogenic mechanism behind Batten disease.This article has an associated First Person interview with the first author of the paper.

Post-translational modifications: How to modulate Rab7 functions.

The small GTPase Rab7 is the main regulator of membrane trafficking at late endosomes. This small GTPase regulates endosome-to-trans Golgi Network trafficking of sorting receptors, membrane fusion of late endosomes to lysosomes, and autophagosomes to lysosomes during autophagy. Rab7, like all Rab GTPases, binds downstream effectors coordinating several divergent pathways. How cells regulate these interactions and downstream functions is not well understood. Recent evidence suggests that Rab7 function can be modulated by the combination of several post-translational modifications that facilitate interactions with one effector while preventing binding to another one. In this review, we discuss recent data on how phosphorylation, palmitoylation and ubiquitination modulate the ability of this small GTPase to orchestrate membrane trafficking at the late endosomes.

The microfluidic laboratory at Synchrotron SOLEIL.

A microfluidic laboratory recently opened at Synchrotron SOLEIL, dedicated to in-house research and external users. Its purpose is to provide the equipment and expertise that allow the development of microfluidic systems adapted to the beamlines of SOLEIL as well as other light sources. Such systems can be used to continuously deliver a liquid sample under a photon beam, keep a solid sample in a liquid environment or provide a means to track a chemical reaction in a time-resolved manner. The laboratory provides all the amenities required for the design and preparation of soft-lithography microfluidic chips compatible with synchrotron-based experiments. Three examples of microfluidic systems that were used on SOLEIL beamlines are presented, which allow the use of X-ray techniques to study physical, chemical or biological phenomena.

In vivo monitoring of the recruitment and activation of AP-1 by Arf1.

AP-1 is a clathrin adaptor recruited to the trans-Golgi Network where it can interact with specific signals found in the cytosolic tail of cargo proteins to incorporate them into clathrin-coated vesicles for trafficking. The small G protein Arf1 regulates the spatiotemporal recruitment of AP-1 and also drives a conformational change favoring an interaction with cargo proteins. A recent crystal structure and in vitro experiments highlighted potential residues mediating the AP-1/Arf1 interaction and the unlocking of the complex. We have used bioluminescence resonance energy transfer (BRET) to study the Arf1/AP-1 interaction and AP-1 conformational changes in vivo. We identified novel residues required for this interaction in addition to those predicted in the crystal structure. We also studied the conformational changes in AP-1 driven by Arf1 in live cells and found that opening of the complex is prerequisite for oligomerization. Using Arf1 knockout cells generated by CRISPR/Cas9, we demonstrated that residue 172 in Arf1 is necessary for AP-1 activation and is required for the efficient sorting of the lysosomal protein prosaposin. We have used BRET to study the in vivo activation of AP-1. The advantages of BRET include expressing full-length proteins in their native environment that have been fully post-translationally modified.

Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking.

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.

Mechanism of insulin resistance in a rat model of kidney disease and the risk of developing type 2 diabetes.

Chronic kidney disease is associated with homeostatic imbalances such as insulin resistance. However, the underlying mechanisms leading to these imbalances and whether they promote the development of type 2 diabetes is unknown. The effect of chronic kidney disease on insulin resistance was studied on two different rat strains. First, in a 5/6th nephrectomised Sprague-Dawley rat model of chronic kidney disease, we observed a correlation between the severity of chronic kidney disease and hyperglycemia as evaluated by serum fructosamine levels (p<0.0001). Further, glucose tolerance tests indicated an increase of 25% in glycemia in chronic kidney disease rats (p<0.0001) as compared to controls whereas insulin levels remained unchanged. We also observed modulation of glucose transporters expression in several tissues such as the liver (decrease of ≈40%, p≤0.01) and muscles (decrease of ≈29%, p≤0.05). Despite a significant reduction of ≈37% in insulin-dependent glucose uptake in the muscles of chronic kidney disease rats (p<0.0001), the development of type 2 diabetes was never observed. Second, in a rat model of metabolic syndrome (Zucker Leprfa/fa), chronic kidney disease caused a 50% increased fasting hyperglycemia (p<0.0001) and an exacerbated glycemic response (p<0.0001) during glucose challenge. Similar modulations of glucose transporters expression and glucose uptake were observed in the two models. However, 30% (p<0.05) of chronic kidney disease Zucker rats developed characteristics of type 2 diabetes. Thus, our results suggest that downregulation of GLUT4 in skeletal muscle may be associated with insulin resistance in chronic kidney disease and could lead to type 2 diabetes in predisposed animals.

CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein.

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.

Adaptive settings for the nearest-neighbor particle tracking algorithm.

BACKGROUND: The performance of the single particle tracking (SPT) nearest-neighbor algorithm is determined by parameters that need to be set according to the characteristics of the time series under study. Inhomogeneous systems, where these characteristics fluctuate spatially, are poorly tracked when parameters are set globally. RESULTS: We present a novel SPT approach that adapts the well-known nearest-neighbor tracking algorithm to the local density of particles to overcome the problems of inhomogeneity. CONCLUSIONS: We demonstrate the performance improvement provided by the proposed method using numerical simulations and experimental data and compare its performance with state of the art SPT algorithms. AVAILABILITY AND IMPLEMENTATION: The algorithms proposed here, are released under the GNU General Public License and are freely available on the web at http://sourceforge.net/p/adaptivespt. CONTACT: javier.mazzaferri@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Analysis of AQP4 trafficking vesicle dynamics using a high-content approach.

Aquaporin-4 (AQP4) is found on the basolateral plasma membrane of a variety of epithelial cells, and it is widely accepted that microtubules play an important role in protein trafficking to the plasma membrane. In the particular case of polarized trafficking, however, most evidence on the involvement of microtubules has been obtained via biochemistry experiments and single-shot microscopy. These approaches have provided essential information, even though they neglect the dynamical details of microtubule transport. In this work, we present a high-content framework in which time-lapse imaging, and single-particle-tracking algorithms were used to study a large number (∼10(4)) of GFP-AQP4-carrying vesicles on a large number of cells (∼170). By analyzing several descriptors in this large sample of trajectories, we were able to obtain highly statistically significant results. Our results support the hypothesis that AQP4 is transported along microtubules, but to our surprise, this transport is not directed straight to the basolateral plasma membrane. On the contrary, these vesicles move stochastically along microtubules, changing direction repeatedly. We propose that the role of microtubules in the basolateral trafficking of AQP4 is to increase the efficiency, rather than determine the specificity of the target.