p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.

Current Methods and Pipelines for Image-Based Quantitation of Nuclear Shape and Nuclear Envelope Abnormalities.

Any given cell type has an associated "normal" nuclear morphology, which is important to maintain proper cellular functioning and safeguard genomic integrity. Deviations from this can be indicative of diseases such as cancer or premature aging syndrome. To accurately assess nuclear abnormalities, it is important to use quantitative measures of nuclear morphology. Here, we give an overview of several nuclear abnormalities, including micronuclei, nuclear envelope invaginations, blebs and ruptures, and review the current methods used for image-based quantification of these abnormalities. We discuss several parameters that can be used to quantify nuclear shape and compare their outputs using example images. In addition, we present new pipelines for quantitative analysis of nuclear blebs and invaginations. Quantitative analyses of nuclear aberrations and shape will be important in a wide range of applications, from assessments of cancer cell anomalies to studies of nucleus deformability under mechanical or other types of stress.

Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway.

Hutchinson-Gilford progeria syndrome (HGPS) is an incurable premature aging disease. Identifying deregulated biological processes in HGPS might thus help define novel therapeutic strategies. Fibroblasts from HGPS patients display defects in nucleocytoplasmic shuttling of the GTP-bound form of the small GTPase Ran (RanGTP), which leads to abnormal transport of proteins into the nucleus. We report that microtubule stabilization in HGPS cells sequestered the nonclassical nuclear import protein Transportin-1 (TNPO1) in the cytoplasm, thus affecting the nuclear localization of its cargo, including the nuclear pore protein NUP153. Consequently, nuclear Ran, nuclear anchorage of the nucleoporin TPR, and chromatin organization were disrupted, deregulating gene expression and inducing senescence. Inhibiting N-acetyltransferase 10 (NAT10) ameliorated HGPS phenotypes by rebalancing the nuclear to cytoplasmic ratio of TNPO1. This restored nuclear pore complex integrity and nuclear Ran localization, thereby correcting HGPS cellular phenotypes. We observed a similar mechanism in cells from healthy aged individuals. This study identifies a nuclear import pathway affected in aging and underscores the potential for NAT10 inhibition as a possible therapeutic strategy for HGPS and perhaps also for pathologies associated with normal aging.

Inhibition of TBC1D5 activates Rab7a and can enhance the function of the retromer cargo-selective complex.

The retromer complex is a vital component of the endosomal protein sorting machinery necessary for sorting into both the endosome-to-Golgi retrieval pathway and also the endosome-to-cell-surface recycling pathway. Retromer mediates cargo selection through a trimeric complex comprising VPS35, VPS29 and VPS26, which is recruited to endosomes by binding to Rab7a and Snx3. Retromer function is linked to two distinct neurodegenerative diseases, Parkinson's disease and Alzheimer's disease and modulating retromer function has been proposed as an avenue to explore for a putative therapy in these conditions. We hypothesised that activating Rab7a to promote the recruitment of retromer to endosomes could positively modulate its activity. Here, we show that inhibition of the GTPase activating protein TBC1D5 can enhance Rab7a activation and lead to a gain of function for retromer.

Analysis of novel endosome-to-Golgi retrieval genes reveals a role for PLD3 in regulating endosomal protein sorting and amyloid precursor protein processing.

The processing of amyloid precursor protein (APP) to the neurotoxic pro-aggregatory Aß peptide is controlled by the mechanisms that govern the trafficking and localisation of APP. We hypothesised that genes involved in endosomal protein sorting could play an important role in regulating APP processing and, therefore, analysed ~ 40 novel endosome-to-Golgi retrieval genes previously identified in a genome-wide siRNA screen. We report that phospholipase D3 (PLD3), a type II membrane protein, functions in endosomal protein sorting and plays an important role in regulating APP processing. PLD3 co-localises with APP in endosomes and loss of PLD3 function results in reduced endosomal tubules, impaired trafficking of several membrane proteins and reduced association of sortilin-like 1 with APP.

Genome-wide RNAi screen reveals a role for multipass membrane proteins in endosome-to-golgi retrieval.

Endosome-to-Golgi retrieval is an essential membrane trafficking pathway required for many important physiological processes and linked to neurodegenerative disease and infection by bacterial and viral pathogens. The prototypical cargo protein for this pathway is the cation-independent mannose 6-phosphate receptor (CIMPR), which delivers lysosomal hydrolases to endosomes. Efficient retrieval of CIMPR to the Golgi requires the retromer complex, but other aspects of the endosome-to-Golgi retrieval pathway are poorly understood. Employing an image-based antibody-uptake assay, we conducted a genome-wide RNAi loss-of-function screen for novel regulators of this trafficking pathway and report ∼90 genes that are required for endosome-to-Golgi retrieval of a CD8-CIMPR reporter protein. Among these regulators of endosome-to-Golgi retrieval are a number of multipass membrane-spanning proteins, a class of proteins often overlooked with respect to a role in membrane trafficking. We further demonstrate a role for three multipass membrane proteins, SFT2D2, ZDHHC5, and GRINA, in endosome-to-Golgi retrieval.

Mutation in VPS35 associated with Parkinson's disease impairs WASH complex association and inhibits autophagy.

Endosomal protein sorting controls the localization of many physiologically important proteins and is linked to several neurodegenerative diseases. VPS35 is a component of the retromer complex, which mediates endosome-to-Golgi retrieval of membrane proteins such as the cation-independent mannose 6-phosphate receptor. Furthermore, retromer is also required for the endosomal recruitment of the actin nucleation promoting WASH complex. The VPS35 D620N mutation causes a rare form of autosomal-dominant Parkinson's disease (PD). Here we show that this mutant associates poorly with the WASH complex and impairs WASH recruitment to endosomes. Autophagy is impaired in cells expressing PD-mutant VPS35 or lacking WASH. The autophagy defects can be explained, at least in part, by abnormal trafficking of the autophagy protein ATG9A. Thus, the PD-causing D620N mutation in VPS35 restricts WASH complex recruitment to endosomes, and reveals a novel role for the WASH complex in autophagosome formation.

Image-based and biochemical assays to investigate endosomal protein sorting.

The sorting of membrane proteins within the endosomal system occurs through a panoply of highly dynamic sequential molecular interactions that together govern many physiologically important processes. A key component of the endosomal protein sorting machinery is the retromer complex. Through two distinct subcomplexes, retromer operates to select cargo for endosome-to-Golgi retrieval and also drives membrane tubule formation. Many accessory proteins associate with retromer to facilitate protein sorting and/or tubule formation. The experience we have gained from studying retromer-mediated endosomal protein sorting and the assays developed and applied in the course of these studies can provide a template for researchers interested in related endosomal trafficking pathways. Herein we describe image-based assays that can be applied to study endosomal protein sorting through the use of antibody-uptake assays in low-, medium-, and high-throughput formats. We additionally detail simple but effective native immunoprecipitation methods that can be employed to identify novel proteins that may interact transiently with a protein of interest within the endosomal pathway.

Inorganic phosphate modulates the expression of the NaPi-2a transporter in the trans-Golgi network and the interaction with PIST in the proximal tubule.

Inorganic phosphate (Pi) homeostasis is maintained by the tight regulation of renal Pi excretion versus reabsorption rates that are in turn modulated by adjusting the number of Pi transporters (mainly NaPi-2a) in the proximal tubules. In response to some hormones and a high dietary Pi content, NaPi-2a is endocytosed and degraded in the lysosomes; however, we show here that some NaPi-2a molecules are targeted to the trans-Golgi network (TGN) during the endocytosis. In the TGN, NaPi-2a interacts with PIST (PDZ-domain protein interacting specifically with TC10), a TGN-resident PDZ-domain-containing protein. The extension of the interaction is proportional to the expression of NaPi-2a in the TGN, and, consistent with that, it is increased with a high Pi diet. When overexpressed in opossum kidney (OK) cells, PIST retains NaPi-2a in the TGN and inhibits Na-dependent Pi transport. Overexpression of PIST also prevents the adaptation of OK cells to a low Pi culture medium. Our data supports the view that NaPi-2a is subjected to retrograde trafficking from the plasma membrane to the TGN using one of the machineries involved in endosomal transport and explains the reported expression of NaPi-2a in the TGN.

Recruitment of the endosomal WASH complex is mediated by the extended 'tail' of Fam21 binding to the retromer protein Vps35.

The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott-Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer-WASH complex interaction occurs through the long unstructured 'tail' domain of the WASH complex-Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.

The cargo-selective retromer complex is a recruiting hub for protein complexes that regulate endosomal tubule dynamics.

The retromer complex is required for the efficient endosome-to-Golgi retrieval of the CIMPR, sortilin, SORL1, wntless and other physiologically important membrane proteins. Retromer comprises two protein complexes that act together in endosome-to-Golgi retrieval; the cargo-selective complex is a trimer of VPS35, VPS29 and VPS26 that sorts cargo into tubules for retrieval to the Golgi. Tubules are produced by the oligomerization of sorting nexin dimers. Here, we report the identification of five endosomally-localised proteins that modulate tubule formation and are recruited to the membrane via interactions with the cargo-selective retromer complex. One of the retromer-interacting proteins, strumpellin, is mutated in hereditary spastic paraplegia, a progressive length-dependent axonopathy. Here, we show that strumpellin regulates endosomal tubules as part of a protein complex with three other proteins that include WASH1, an actin-nucleating promoting factor. Therefore, in addition to a direct role in endosome-to-Golgi retrieval, the cargo-selective retromer complex also acts as a platform for recruiting physiologically important proteins to endosomal membranes that regulate membrane tubule dynamics.

Differential regulation of the renal sodium-phosphate cotransporters NaPi-IIa, NaPi-IIc, and PiT-2 in dietary potassium deficiency.

Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate (P(i)) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-P(i) cotransporter NaPi-IIa, but that the activity, diffusion, and clustering of NaPi-IIa could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney Int 60: 694-704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160-49171, 2004). Here we investigated the role of the renal Na-P(i) cotransporters NaPi-IIc and PiT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-IIc protein abundance in proximal tubular BBM and in NaPi-IIc mRNA. In addition, we documented the presence of a third Na-coupled P(i) transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-IIc in K deficiency: in control rats, NaPi-IIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPi-IIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-IIc and also PiT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations.

Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi IIa transporter to the trans-Golgi.

The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH(2) end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAP17 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa.

Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy.

With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Forster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment.

Shank2E binds NaP(i) cotransporter at the apical membrane of proximal tubule cells.

Proteins expressing postsynaptic density (PSD)-95/Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaP(i) cotransporter (NaP(i)-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaP(i)-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E co-immunoprecipitated with NaP(i)-IIa from rat renal cortex tissue and HA-NaP(i)-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaP(i)-IIa and truncation of the COOH-terminal TRL motif from NaP(i)-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaP(i)-IIa interaction occurred directly between the two proteins. NaP(i)-IIa activity is regulated by moderating its abundance in the apical membrane. High-P(i) conditions induce NaP(i)-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-P(i) conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaP(i)-IIa via PDZ interactions, and undergoes P(i)-induced internalization.

Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells.

We previously showed an inverse correlation between membrane cholesterol content and Na-P(i) cotransport activity during the aging process and adaptation to alterations in dietary P(i) in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P(i) cotransport activity and apical membrane Na-P(i) protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with beta-methyl cyclodextrin (beta-MCD) resulted in a significant increase in Na-P(i) cotransport activity accompanied by a moderate increase in apical membrane Na-P(i) protein abundance and no alteration of total cellular Na-P(i) protein abundance. Conversely, acute cholesterol enrichment achieved with beta-MCD/cholesterol resulted in a significant decrease in Na-P(i) cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P(i) protein abundance. In contrast, chronic cholesterol depletion, achieved by growing cells in lipoprotein-deficient serum (LPDS), resulted in parallel and significant increases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Cholesterol depletion also resulted in a significant increase in membrane lipid fluidity and alterations in lipid microdomains as determined by laurdan fluorescence spectroscopy and imaging. Chronic cholesterol enrichment, achieved by growing cells in LPDS followed by loading with low-density lipoprotein, resulted in parallel and significant decreases in Na-P(i) cotransport activity and apical membrane and total cellular Na-P(i) protein abundance. Our results indicate that in OK cells acute and chronic alterations in cholesterol content per se modulate Na-P(i) cotransport activity by diverse mechanisms that also include significant interactions of Na-P(i) protein with lipid microdomains.

Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.