Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1-2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP-ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP-ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP-ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.

The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

BACKGROUND

The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.

RESULTS

Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells.

CONCLUSIONS

We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.

The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

Rab5 is a GTPase that regulates early endosome fusion. Its GTPase domain crystal structure is reported here at 1.05 A resolution in complex with a GTP-analog molecule. It provides the highest resolution three-dimensional model so far obtained for proteins from the Ras-like GTPase family. This study allows extension of structural examination of the GTPase machinery as well as of high-resolution protein structures in general. For example, a buried water-molecule network was observed underneath the switch regions, which is consistent with the functional roles of these regions in the molecular-switching process. Furthermore, residues of multiple conformation and clustered distribution of anisotropic thermal motions of the protein molecule may have general implications for the function of Ras-like GTPases.

We have previously shown that Xenopus rabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J. 16, 6182-6191). In this report we demonstrate that caspase-dependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD(379) and DESD(438)) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coil-forming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5.

The two early endosome-associated rab GTP-binding proteins, rab4 and rab5, are suggested to regulate endocytosis. In this report, we examined post-translational processing and membrane association of the two rab proteins. Human rab4 and rab5 were expressed in chicken embryo fibroblasts using a Sindbis virus expression vector. Cells were labeled with either [35S]methionine or [3H]mevalonolactone. Cell lysates were immunoprecipitated with antisera specific for rab4 and rab5, respectively, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that both rab4 and rab5 contained at least three forms: the precursor form, the isoprenylated intermediate, and the isoprenylated mature form of faster mobility. The rab5 intermediate comigrated with the precursor form, whereas the rab4 intermediate migrated slightly faster than the precursor form. The intermediate form of rab4, but not rab5, accumulated in the presence of a chymotrypsin-like protease inhibitor (N-acetyl Tyr ethyl ester), suggesting that proteolysis was required for generation of the mature form. Furthermore, the intermediate and mature forms of rab4, but not rab5, were carboxyl-methylated as demonstrated by incorporation of alkali-labile counts from [methyl-3H]methionine. Membrane association of the distinct rab4 and rab5 forms was examined by subcellular fractionation and Triton X-114 partitioning. The precursor forms were found in the cytosol and partitioned into the aqueous phase. The mature forms were membrane-associated and partitioned into the detergent phase. Unexpectedly, the isoprenylated intermediate forms of both rab4 and rab5 partitioned exclusively into the aqueous phase. Taken together, the data indicate that the entire post-translational processing, which includes isoprenylation, carboxyl methylation (rab4), and possibly proteolysis, confers the competency for membrane association of rab4 and rab5.

Wortmannin reportedly induces the formation of enlarged cytoplasmic endosomes. Such vesicles were observed in a definite time window after wortmannin treatment (250 nM) in HEK-293 cells stably expressing a B2R (B2 receptor)--green fluorescent protein conjugate and other cell types. The alternative PI3K (phosphoinositide 3-kinase) inhibitor LY 294002 (100 microM) and a dominant-negative form of the enzyme (p85alpha DeltaiSH2) induce a more modest vesicle enlargement. PI3K inhibition by drugs did not affect agonist-induced [3H]arachidonate release. The wortmannin-induced formation of giant endosomes also involves Rab5 activity, since a dominant-negative form of this GTPase (Rab5 S34N) partially inhibits the wortmannin effect and a constitutively active form of Rab5 (Rab5 Q79L) induces the formation of enlarged endosomes. Moreover, agonist stimulation targeted B2R-green fluorescent protein towards the periphery of the giant vesicles and led to partial receptor degradation only in wortmannin-treated cells. Receptor degradation was decreased by protease inhibitors and by bafilomycin A1, a drug that inhibits lysosome function. Accumulation of fluorescent material inside the enlarged endosomes was observed in cells treated with bafilomycin A1, wortmannin and an agonist. [3H]Bradykinin binding was decreased in HEK-293 cells treated with both wortmannin and the agonist, but not with either separately. Furthermore, a wortmannin-induced functional down-regulation of B2R was observed in rabbit jugular veins after repeated agonist stimulation (contractility assay). This is the first report of a G-protein-coupled receptor down-regulation induced by an alteration of its usual routing in the cell. These results suggest that both PI3K and Rab5 influence B2R intracellular trafficking.

Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport. We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro. Here, we show that this process depends on the rab5 N-terminal domain. Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide. Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions. Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol. Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments. Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus. A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay. A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects. Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery.

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.

BACKGROUND

We address the task of parameter estimation in models of the dynamics of biological systems based on ordinary differential equations (ODEs) from measured data, where the models are typically non-linear and have many parameters, the measurements are imperfect due to noise, and the studied system can often be only partially observed. A representative task is to estimate the parameters in a model of the dynamics of endocytosis, i.e., endosome maturation, reflected in a cut-out switch transition between the Rab5 and Rab7 domain protein concentrations, from experimental measurements of these concentrations. The general parameter estimation task and the specific instance considered here are challenging optimization problems, calling for the use of advanced meta-heuristic optimization methods, such as evolutionary or swarm-based methods.

RESULTS

We apply three global-search meta-heuristic algorithms for numerical optimization, i.e., differential ant-stigmergy algorithm (DASA), particle-swarm optimization (PSO), and differential evolution (DE), as well as a local-search derivative-based algorithm 717 (A717) to the task of estimating parameters in ODEs. We evaluate their performance on the considered representative task along a number of metrics, including the quality of reconstructing the system output and the complete dynamics, as well as the speed of convergence, both on real-experimental data and on artificial pseudo-experimental data with varying amounts of noise. We compare the four optimization methods under a range of observation scenarios, where data of different completeness and accuracy of interpretation are given as input.

CONCLUSIONS

Overall, the global meta-heuristic methods (DASA, PSO, and DE) clearly and significantly outperform the local derivative-based method (A717). Among the three meta-heuristics, differential evolution (DE) performs best in terms of the objective function, i.e., reconstructing the output, and in terms of convergence. These results hold for both real and artificial data, for all observability scenarios considered, and for all amounts of noise added to the artificial data. In sum, the meta-heuristic methods considered are suitable for estimating the parameters in the ODE model of the dynamics of endocytosis under a range of conditions: With the model and conditions being representative of parameter estimation tasks in ODE models of biochemical systems, our results clearly highlight the promise of bio-inspired meta-heuristic methods for parameter estimation in dynamic system models within system biology.

Oxidative stress is considered important for the pathogenesis of Alzheimer disease (AD), which is characterized by the formation of senile plaques rich in amyloid beta-protein (Abeta). Abeta cytotoxicity has been found dependent on lysosomes, which are abundant in AD neurons and are shown to partially co-localize with Abeta. To determine whether oxidative stress has any influence on the relationship between lysosomes and Abeta1-42 (the most toxic form of Abeta), we studied the effect of hyperoxia (40% versus 8% ambient oxygen) on the intracellular localization of Abeta1-42 (assessed by immunocytochemistry) in retinoic acid differentiated SH-SY5Y neuroblastoma cells maintained in serum-free OptiMEM medium. In control cells, Abeta1-42 was mainly localized to small non-lysosomal cytoplasmic granules. Only occasionally Abeta1-42 was found in large (over 1 microm) lysosomal-associated membrane protein 2 positive vacuoles, devoid of the early endosomal marker rab5. These large Abeta1-42 -containing lysosomes were not detectable in the presence of serum (known to suppress autophagy), while their number increased dramatically (up to 24-fold) after exposure of cells to hyperoxia during 5 days. Activation of autophagy by hyperoxia was confirmed by transmission electron microscopy. Furthermore, an inhibitor of autophagic sequestration 3-methyladenine prevented the accumulation of Abeta1-42 -positive lysosomes due to hyperoxia. In parallel experiments, intralysosomal accumulation of Abeta1-40 following oxidative stress has been found as well. The results suggest that Abeta can be autophagocytosed and its accumulation within neuronal lysosomes is enhanced by oxidative stress.

Endocytic pathway abnormalities were previously observed in brains affected with Alzheimer's disease (AD). To clarify the pathological relevance of these abnormalities to assembly of amyloid beta-protein (Abeta), we treated PC12 cells with chloroquine, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment induced accumulation of GM1 ganglioside (GM1) in Rab5-positive enlarged early endosomes and on the cell surface. Notably, an increase in GM1 level on the cell surface was sufficient to induce Abeta assembly. Our results suggest that endocytic pathway abnormalities in AD brain induce GM1 accumulation on the cell surface, leading to amyloid fibril formation in brain.

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.

Recycling of 46,000 M(r) mannose 6-phosphate receptor (MPR 46) was investigated by microinjection of Fab fragments against small epitopes within the cytoplasmic domain of the receptor. Fab fragments against the peptide 43-47 (Ala-Tyr-Arg-Gly-Val) efficiently blocked return of MPR 46 to the TGN. Antibody-induced redistribution resulted in accumulation of MPR 46 within an endosomal compartment, from which it recycled to the plasma membrane. Rab5 and rab7, markers for early and late endosomes, respectively, were not detectable in the compartment of redistributed MPR 46, suggesting that it represents a specialized endosomal subcompartment. The bulk of redistributed MPR 46 did not colocalize with endocytosed fluid-phase marker, suggesting that it accumulates at a site where MPR 46 has been segregated from endocytosed material, which is destined for transport to lysosomes. Peptide 43-47 contains a tyrosine (residue 44) which has been shown earlier to be part of an internalization signal for MPR 46 (Johnson, K. F., W. Chan, and S. Kornfeld. 1990. Proc. Natl. Acad. Sci. USA. 87:10010-10014). The role of tyrosine residue 44 as part of a putative multifunctional sorting signal is discussed.

Endosomal sorting complexes required for transport (ESCRTs) regulate a key sorting step of protein trafficking between endosomal compartments in lysosomal degradation. Interestingly, mutations in charged multivesicular body protein 2B (CHMP2B), which is a core subunit of ESCRT-III, have been identified in some neurodegenerative diseases. However, the cellular pathogenesis resulting from CHMP2B missense mutations is unclear. Furthermore, little is known about their functional analysis in post-mitotic neurons. In order to examine their cellular pathogenesis, we analyzed their effects in the endo-lysosomal pathway in post-mitotic neurons. Interestingly, of the missense mutant proteins, CHMP2B(T104N) mostly accumulated in the Rab5- and Rab7-positive endosomes and caused delayed degradation of EGFR as compared to CHMP2B(WT). Furthermore, CHMP2B(T104N) showed less association with Vps4 ATPase and was avidly associated with Snf7-2, a core component of ESCRT-III, suggesting that it may cause defects in the process of dissociation from ESCRT. Of the missense variants, CHMP2B(T104N) caused prominent accumulation of autophagosomes. However, neuronal cell survival was not dramatically affected by expression of CHMP2B(T104N). These findings suggested that, from among the various missense mutants, CHMP2B(T104N) was associated with relatively mild cellular pathogenesis in post-mitotic neurons. This study provided a better understanding of the cellular pathogenesis of neurodegenerative diseases associated with various missense mutations of CHMP2B as well as endocytic defects.

Appl1 (Adaptor protein containing pleckstrin homology [PH], phosphotyrosine binding [PTB], and Leucine zipper motifs) is an adaptor that participates in cell signaling by interacting with various signaling molecules including Akt, PI3-kinase (PI3K), Rab5, adiponectin receptor, and TrkA. By using RNA knockdown technology, Appl1 has been implicated in zebrafish development and murine glucose metabolism. To investigate the unambiguous role of Appl1 in vivo, we generated a knockout mouse in which exon1 of the Appl1 gene was disrupted using gene trap methodology. Homozygous Appl1 knockout mice with ubiquitous loss of Appl1 protein expression were viable, grossly normal, and born at expected Mendelian ratios. Moreover, activation of Akt and the downstream effecter Gsk3β was unaffected in vivo. We next performed glucose and insulin tolerance tests and found that glucose metabolism is normal in Appl1-null mice. We also tested the effect of Appl1 loss on Akt signaling in T cells, because we discovered that Appl1 strongly interacts with the p110β subunit of PI3K in T lymphocytes. However, such interaction was found to be dispensable for Akt signaling in thymic T cells and T-cell development. Moreover, Appl1 loss did not affect DNA synthesis in cultured thymocytes, although loss of Appl1 was associated with a slight increase in ConA-stimulated splenic T-cell viability/proliferation. Collectively, our findings indicate that Appl1 is dispensable for Akt signaling in vivo and T-cell differentiation.

Given that cancer stem cells (CSC) play a key role in drug resistance and relapse, targeting CSCs remains promising in cancer therapy. Here we show that RAB5/7, which are involved in the endolysosomal pathway, play key roles in the maintenance of CSC survival via regulation of the mitophagic pathway. Inhibition of RAB5/7 efficiently eliminated colorectal CSCs and disrupted cancer foci. In addition, we identified mefloquine hydrochloride, an antimalarial drug, as a novel RAB5/7 inhibitor and promising colorectal CSC-targeting drug. Endolysosomal RAB5/7 and LAMP1/2 mediated parkin-dependent mitochondrial clearance and modulated mitophagy through lysosomal dynamics. In a patient-derived xenograft (PDX) model of colon cancer, treatment with mefloquine resulted in suppression of mitophagic PINK1/PARKIN and increased mitochondrial disorder and mitochondria-induced apoptosis without apparent side effects. These results suggest that the combination of mefloquine with chemotherapeutic agents in the PDX model potentially disrupts the hierarchy of colorectal cancer cells and identify endolysosomal RAB5/7 and LAMP1/2 as promising therapeutic targets in CSCs. SIGNIFICANCE: These findings show that endosomal/lysosomal RAB5 and RAB7, which regulate mitophagy, are essential for the survival of colon cancer stem cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1426/F1.large.jpg.

Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biological problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-associated infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The experimental results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-containing vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-positive endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.

In this study the uptake of tissue factor (TF)-positive microparticles by endothelial cells and the recycling of the TF component were examined. Human dermal blood endothelial cells (HDBEC) were incubated with microparticles derived from cancer cell lines for up to 6 hours. Measurement of HDBEC cell surface TF antigen revealed two distinct peaks at 30 and 180-240 minutes post-incubation with TF-positive, but not TF-deficient microparticles. However, only the second peak was concurrent with high TF activity as determined by a chromogenic thrombin-generation assay. Annexin V-labelling of HDBEC showed phosphatidylserine exposure following 90 minutes incubation with microparticles, which explains the high TF activity associated with the second antigen peak. Analysis of TF mRNA levels revealed no de novo expression of TF mRNA in response to microparticles, and pre-incubation of cells with cycloheximide did not prevent the appearance of TF. However, blocking endocytosis with a dynamin inhibitor prolonged the disappearance and prevented the reappearance of TF antigen on the cell surface. Incubation of HDBEC with microparticles containing TF-GFP revealed the early co-localisation of TF with Rab4 and Rab5, followed by co-localisation with the late endosomal/trans-Golgi network marker Rab9, and the recycling endosome marker Rab11. siRNA-mediated suppression of Rab11 reduced the reappearance of TF on the cell surface. These data suggest a mechanism by which TF-containing microparticles are internalised by endothelial cells and the TF moiety recycled to the cell surface. Together with the exposure of phosphatidylserine, this is capable of inducing a substantial increase in the procoagulant potential of the surface of endothelial cells.

The metabolic signatures of cancer cells are often associated with elevated glycolysis. Pharmacological (PFK158 treatment) and genetic inhibition of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a critical control point in the glycolytic pathway, decreases glucose uptake, ATP production, and lactate dehydrogenase activity and arrests malignant pleural mesothelioma (MPM) cells in the G0/G1 phase to induce cell death. To overcome this nutrient stress, inhibition of PFKFB3 activity led to an escalation in endoplasmic reticulum (ER) activity and aggravated ER stress mostly by upregulating BiP and GADD153 expression and activation of the endocytic Rac1-Rab5-Rab7 pathway resulting in a unique form of cell death called "methuosis" in both the sarcomatoid (H28) and epithelioid (EMMeso) cells. Transmission electron microscopy (TEM) analysis showed the formation of nascent macropinocytotic vesicles, which rapidly coalesced to form large vacuoles with compromised lysosomal function. Both immunofluorescence microscopy and co-immunoprecipitation analyses revealed that upon PFKFB3 inhibition, two crucial biomolecules of each pathway, Rac1 and Calnexin interact with each other. Finally, PFK158 alone and in combination with carboplatin-inhibited tumorigenesis of EMMeso xenografts in vivo. Since most cancer cells exhibit an increased glycolytic rate, these results provide evidence for PFK158, in combination with standard chemotherapy, may have a potential in the treatment of MPM.

Intrinsic apoptosis involves BH3-only protein activation of Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP). Consequently, cytochrome c is released from the mitochondria to activate caspases, and Smac (second mitochondria-derived activator of caspases) to inhibit XIAP-mediated caspase suppression. Dysfunctional mitochondria can be targeted for lysosomal degradation via autophagy (mitophagy), or directly through mitochondria-derived vesicle transport. However, the extent of autophagy and lysosomal interactions with apoptotic mitochondria remains largely unknown. We describe here a novel pathway of endolysosomal processing of mitochondria, activated in response to canonical BH3-only proteins and mitochondrial depolarization. We report that expression of canonical BH3-only proteins, tBid, BimEL, Bik, Bad, and mitophagy receptor mutants of atypical BH3-only proteins, Bnip3 and Bnip3L/Nix, leads to prominent relocalization of endolysosomes into inner mitochondrial compartments, in a manner independent of mitophagy. As an upstream regulator, we identified the XIAP E3 ligase. In response to mitochondrial depolarization, XIAP actuates Bax-mediated MOMP, even in the absence of BH3-only protein signaling. Subsequently, in an E3 ligase-dependent manner, XIAP rapidly localizes inside all the mitochondria, and XIAP-mediated mitochondrial ubiquitylation catalyses interactions of Rab membrane targeting components Rabex-5 and Rep-1 (RFP-tagged Rab escort protein-1), and Rab5- and Rab7-positive endolysosomes, at and within mitochondrial membrane compartments. While XIAP-mediated MOMP permits delayed cytochrome c release, within the mitochondria XIAP selectively signals lysosome- and proteasome-associated degradation of its inhibitor Smac. These findings suggest a general mechanism to lower the mitochondrial apoptotic potential via intramitochondrial degradation of Smac.